Epidemiologic investigation of pathogenic Escherichia coli in domestic dogs in Nanchong area

Epidemiologic investigation of pathogenic Escherichia coli in domestic dogs in Nanchong area | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Epidemiologic investigation of pathogenic Escherichia coli in domestic dogs in Nanchong area Bangyuan Wu, Kai Li, Aifei Du, Shunjie Tang, Shaohua Feng, Shibin Yuan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7708388/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pathogenic Escherichia coli is a zoonotic bacterial pathogen that causes significant losses to the farming industry and threatens public health safety. Dogs, as companion animals, are closely intertwined with human life and health. Therefore, heightened attention to pathogenic E. coli in canines is particularly crucial. This study investigated the infection and incidence of canine pathogenic E. coli disease in Nanchong of Sichuan province in China. 3229 diseased dogs from 2020 to 2021, according to different seasons, gender, age, breed, and immunization status, were analyzed. Meanwhile, pathogenic E. coli was isolated and identified from the fecal samples of the sick dogs, and the virulence factors and drug resistance of the strains were also analyzed. The study identified 129 cases of pathogenic E. coli infections in dogs from 2020 to 2021, accounting for 4% of the total (129/3229). compared to factors such as season, sex, and immune status, there is a significant difference in the infection rates between different age groups of dogs (P=0.029). Canine pathogenic E. coli can be infected throughout the year, The study found that the highest rate of pathogenic E. coli infections in dogs occurred during spring, with summer having the second-highest rate, followed by winter. The infection rate of males was higher than that of females, the infection rate of juvenile dogs was higher than that of adult dogs, and the infection rate of small dogs was higher than that of medium-sized and large dogs. In addition, all 27 pathogenic E. coli strains isolated from diverse geographical regions demonstrated antibiotic susceptibility, with the lowest sensitivity to tetracycline at 60%, followed by amoxicillin, streptomycin, and butyl carbamate, with a sensitivity of 80%. The results of whole gene sequencing showed that the main virulence factors were Fimbriae, LPS, Brk, and the canine pathogenic E. coli in the Nanchong area had developed resistance to some antibiotics. Although the infection rate of pathogenic E. coli in canines was relatively low in Nanchong, it remains essential to advance research on its pathogenic characteristics, antimicrobial resistance patterns, and control measures. Concurrently, clinical antimicrobial use must be standardized, immunity in juvenile dogs enhanced, and environmental hygiene management within breeding facilities strengthened. Dog Drug resistance Epidemiology Toxicity factors Nanchong Pathogenic Escherichia coli Whole genome sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Pathogenic E. coli is a significant safety hazard in different areas of pig, chicken, and duck farming[1], and its features, including difficult to cure, high mortality rate, and very easy to recur, make it a persistent challenge in livestock production [2]. Animals infected with pathogenic E. coli will cause different body inflammation [3], such as urethritis [4], gastroenteritis [5], umbilical cord infection and vitellicle infection [6]. It is essential to consider the antimicrobial resistance (AMR) and concurrent infections when treating E. coli . In addition, the endotoxin released by pathogenic E. coli should also pay attention to [7], which is highly toxic and can cause fever [8], sepsis, toxemia [9], pericarditis [10], nephritis [11], and even induce to death [12]. The initial symptoms of canine E. coli disease are not obvious [13], and it is not easy to cause the attention of dog owners. In the late stage, the sick dog will present depressed, loss of appetite or complete abolition, the severe dehydration phenomenon, poor skin elasticity, eye sockets sunken, limb weakness or body temperature drop [14], and will quickly occur diarrhea, discharge viscous yellowish-white, yellowish-green, or greenish dilute feces, emitting an unpleasant Fishy odor [15]. Pathogenic E. coli infections in puppies often result in diarrhea that produces air bubbles and food residues due to lack of digestion, causing secondary fecal contamination of body surfaces (tail, hind legs, anus). Pathogenic E. coli invades the host via the oral or nasal cavity, evades antimicrobial defenses, and proliferates in the small intestine. It colonizes the intestinal mucosa through the bacterium's colonization factor (pilus or adhesins) [16], which binds to the cells' specific receptors on the mucous membrane's surface, causing inflammation. Pathogenic E. coli can produce endotoxin [17], enterotoxin [18], and colistin [19]. When pathogenic E. coli disintegrates, it releases lipopolysaccharides, and the lipid A component can cause endotoxemia [20]. Enteroproducing virulent E. coli can infect epithelial cells of the small intestine, colonize and proliferate on the cells, and produce enterotoxins [21]. LT and ST as two important enterotoxins; LT (Heat-Labile Enterotoxin) activates adenylyl cyclase in intestinal epithelial cells, increasing cAMP production, which causes hypersecretion, diarrhea, and dehydration[22]. ST (Heat-Stable Enterotoxin) activates guanylyl cyclase (cGMP) in ileal epithelial cells, causing secretory diarrhea; many E. coli produce colicin V (ColV) plasmids[23, 24], This is linked to the bacterium’s ability to cause sepsis. Additionally, specific toxin-producing E. coli (ETEC) can invade and damage intestinal mucosa cells, with invasiveness related to the presence of a plasmid [25]. Ruminants such as sheep, deer, and cattle are the primary hosts of E. coli [26], and the range of the animals and complex environmental influences can cause E. coli migration. In the study of the mechanism of E. col i invasion of soil and its interactions with indigenous microorganisms, it has been found that pathogenic E. coli forms a complex migratory chain with the environment [27], thus threatening the development of the farming industry and even to the health of human beings. A mount of E. coli exists in the feces of infected animals, and according to the survival characteristics of E. coli [28], it can survive for a long time in the adapted environment. The feces of animals infected with pathogenic E. col i migrate through the irrigation of farmland, flies, birds, rainfall flushing, and groundwater flow, forming a closed-loop contamination of food [29] and water [30], which threatens the safety of public health. Therefore, managing the scope of activities of feeding animals is very important for preventing and controlling pathogenic E. coli infection. Moreover, Pathogenic E. coli is a zoonotic disease [31]; the transmission is mainly through food, water, and intimate contact [32], of which food transmission is the primary way. However, due to the simple transmission pathway, E. coli is always a threat to public health safety and the development of animal husbandry. Though the pathogenic E. coli infection situation has improved, there is still a risk of infection outbreaks, especially in dog farms and pet dog breeding [33]. In northeast China, the infection of pathogenic E. coli infections in dogs is as high as 63.75% [34], a statistic that reveals a serious challenge to canine health in the region. In the veterinary and public health fields, high infection rates can lead to a decline in the health of canine populations [35], affecting their quality of life and productivity and highlighting the potential risk of transmission of zoonotic diseases to human health, such as urinary tract infections, nephritis [36]. Nanchong, the second most populous city in Sichuan China, has more families of pet dogs, farm dogs, and stray dogs, and a wide range of activities of these dogs will produce secondary pollution. Hence, the infection rate has been high for a long time. In the present study, the infected with pathogenic E. coli dogs were analyzed through the different seasons, different breeds of dogs, different genders, different age incidence in the Nanchong region, the collection of feces from typical diseased dogs, and the isolation of identified Pathogenic E. coli was collected, and the isolation and identification of pathogenic E. coli , drug sensitivity test, and genotyping were carried out to obtain the corresponding rules, which can be used as the reference data for the prevention of pathogenic E. coli infections in animal husbandry, prevention and control of the northeastern region of Sichuan. This study establishes Nanchong as a sentinel surveillance site to reduce pathogenic E. coli infection prevalence in canine populations through molecular epidemiological characterization of virulence determinants and antimicrobial resistance patterns, thereby developing targeted biosecurity interventions for effective zoonotic transmission control. 2. Materials and Methods 2.1 Sample and data collection The case data for this study were obtained from The Kawakita Animal Hospital in Nanchong City. The hospital was established in 2013, a specialized and well-equipped veterinary institution for dogs and cats, approved by the Nanchong Municipal Animal Husbandry and Veterinary Medicine Department. It focuses on the diagnosis and treatment of pet diseases. Data collection was conducted from January 2020 to December 2021. During this period, a total of 3,229 canine cases were diagnosed, with more than 600 owners contacted via randomized telephone follow-up. The consultation area covered various districts within Nanchong City, including Shunqing District, Xichong County,Jialing District, Gaoping District, Peng’an County, Yingshan County, and Langzhong City, as well as neighboring cities such as Dazhou, Suining, and Chengdu. Samples were grouped by geographic region: Group A (Shunqing District), Group B (Gaoping District), and Group C (Jialing District). Based on the statistical distribution of pathogenic E. coli cases in Nanchong, representative cases were selected from each group for sampling to ensure geographical representation. To account for seasonal variation, in Nanchong City, a follow-up survey of diseased dogs was conducted throughout the four seasons across various districts and counties(spring, summer, autumn, and winter) If a sampled dog recovers, the next eligible dog within the vicinity is selected to maintain full coverage of Nanchong’s districts and counties. Each dog was continuously observed until fecal discharge. Feces were collected aseptically into sterile containers, with the outer layer discarded using sterile swabs. The inner fecal layer was placed into cryotubes, stored in a portable dry ice refrigerator, and transferred to the laboratory for preservation at -80℃ (Yuan et al., 2024). Specifically, 13 samples were collected from Shunqing District, 7 from Xichong County, 7 from Gaoping District, and 3 from Jialing District. 2.2 Isolation, Genotyping, and Drug Sensitivity Testing of Pathogenic E. coli Isolation and identification of strains are suspected pathogenic E. coli strains were isolated from fecal samples through enrichment culture, followed by purification using repeated streaking, and stored at -80℃. Samples were processed by Shanghai Biotechnology Company for strain identification and full-genome sequencing. This allowed for analysis of phylogenetic relationships, annotation of virulence factors, and assessment of antibiotic resistance genes. Bacterial culture and purification strain proliferation culture: Luria-Bertani (LB) broth was prepared using yeast extract (Beijing Auboxing Biotechnology Co., Ltd.) and tryptone (Qingdao HaiBo Biotechnology Co., Ltd.). Approximately 1-2 g of fecal sample was inoculated and incubated at 37℃ for 24 h. Purification culture: MacConkey agar (Qingdao HaiBo Biotechnology Co., Ltd.) was used for isolation. Serial dilutions (10^1 to 10^5) of the samples were plated using a sterile spreader. Plates were incubated at 37℃ for 24 h. Single colonies were repeatedly purified for three generations. Strain Preservation: Purified colonies were mixed with 40% sterilized glycerol and stored in EP tubes at -20℃ (Ribeiro-Almeida et al., 2024). 2.3 Genome Extraction, PCR Amplification, and Sequencing Genomic DNA extraction: DNA was extracted using the BigDye Sequencing Reaction Kit (SK1201-UNIQ-10) following manufacturer instructions. PCR amplification: PCR was performed using the BigDye Sequencing Reaction Kit (4337455, Shanghai Sangon). The thermal cycle was as follows: initial denaturation at 94℃ for 5 min, followed by 35 cycles of 94℃ for 30 s, 55℃ for 35 s, and 72℃ for 1 min, with a final extension at 72℃ for 8 min. Gel electrophoresis and purification: PCR products were analyzed via agarose gel electrophoresis. Desired DNA bands were excised and purified according to the instructions of SK1131. Sequencing primers: M13-47 primer (AGGGTTTTCCCAGTCACG) was used for sequencing (Supplemental Data Table 1). Sequencing reaction and processing: PCR sequencing was performed in PCR tubes placed on ice. After DNA precipitation with sodium acetate and ethanol, centrifugation was conducted, followed by washing with 70% ethanol. DNA was dried under vacuum, resuspended in TSR buffer, heat-denatured, and cooled on ice before sequencing (supplemental data Table 2). Capillary electrophoresis was conducted with correct positioning and automatic gel loading. Pre-electrophoresis was done at 1.2 kV for 5 min, followed by electrophoresis at 7.5 kV for 2 h. Sequence analysis was performed automatically. 2.4 Annotation and Phylogenetic Analysis VFDB (Virulence Factor Database): The protein sequences of predicted genes were aligned using BLAST against the VFDB, which contains 32,638 genes related to virulence factors across 2,611 entries, all supported by experimental evidence. CARD (Comprehensive Antibiotic Resistance Database): This database contains 2,359 sequences and 3,567 Antibiotic Resistance Ontology (ARO) terms. Sequences were aligned using BLAST to identify resistance-related annotations. 16S rRNA gene analysis: Highly conserved and present in all bacteria, the 16S rRNA gene sequences were compared with NCBI’s 16S database using BLAST. The top 30 hits were selected for phylogenetic analysis. These sequences were then aligned and used to construct a phylogenetic tree. 2.5 Drug Sensitivity Test Methods The drug sensitivity of pathogenic E. coli was tested using the Disk Diffusion Test (supplemental data Table 3). MH medium (028050, MH agar Hangzhou Binhe Microbiology Reagent Co., Ltd.) was inoculated with the bacteria, and a sterile cotton swab was used to coat the medium uniformly. Sensitive paper sheets (921171, detection of drug sensitivity tablets, Changde Beekman Biotechnology Co., Ltd.) were placed on the plate, ensuring a center distance of at least 24 mm. After incubating at 37℃ for 24 h, the inhibition circles were measured and classified as resistant (R), moderately sensitive (I), or sensitive (S) based on their size. A larger circle indicated greater sensitivity and less resistance, while a smaller or absent circle indicated resistance. Interpretation criteria for the diameter of the circle of inhibition of commonly used drug sensitizers for Enterobacteriaceae. The diameter of the circle of inhibition reflects the antimicrobial activity of different antibiotics against bacteria at specific concentrations. The experiment tested the antimicrobial activity of 17 antibiotics most commonly used in clinical practice, such as ampicillin, amoxicillin, and cefazolin, against bacteria at specific concentrations, which were categorized as resistant (≤13 mm), moderately sensitive (14-20 mm), and sensitive (≥21 mm), respectively (Humphries et al., 2018). 2.5 Methods of statistical analysis of survey data and Data analysis EXCEL was used to statistically organize the data to obtain the incidence rate of pathogenic E. coli-infected dogs, and the formula was calculated: ① different seasonal incidence rate (%) = the number of different seasonal incidences / the total number of cases of pathogenic E. coli disease clinic × 100% ② different breeds of dogs morbidity (%) = the number of different breeds of dogs onset / the total number of cases of pathogenic E. coli disease clinic × 100% ③ Incidence rate of dogs of different ages (%) = the number of dogs of different ages with disease / total number of cases of pathogenic E. coli disease × 100% ④ Incidence rate of dogs of different genders (%) = number of dogs of different genders/total number of diagnosed cases of pathogenic E. coli disease×100%. ⑤ Immunized or not (%)=Number of immunized or not/total number of cases of pathogenic E. coli disease×100%. The canine cases were categorized by age: 1-60 days old for suckling pup, 2 months to 12 months old for young dogs, 13 months old-84 months old for adult dogs, and 84 months old and above for senior dogs. An ANOVA analysis was conducted on dogs infected with pathogenic E. coli , examining the effects of different seasons, ages, genders, and immunity acquisition or loss status following immunization. This analysis was performed using SPSS 26.0 software. A p-value of < 0.05 determined statistical significance. 3. Results 3.1 Morbidity in consulting dogs As illustrated in Figure 1, a total of 3,229 sick dogs were collected from January 2020 to December 2021 at The Kawakita Animal Hospital in Nanchong. The diseases were categorized into specific groups based on their clinical presentation. Among the total number of 3,229 sick dogs, gastrointestinal diseases were the most prevalent, with 733 cases, accounting for 22.7% of the total number of dogs examined. This was followed by skin diseases, which affected 505 dogs (15.64%). Infectious diseases were the third most common, with 384 affected dogs (11.89%), while genitourinary diseases were recorded in 300 dogs, representing 9.29% of the total cases. These results underscore the significance of gastrointestinal and dermatological conditions as the most frequent health issues observed in the population of sick dogs. 3.2. Annual incidence of pathogenic E. coli In 2020, a total of 1,837 sick dogs were admitted, among which 71 were diagnosed with pathogenic infections, resulting in an annual infection rate of 3.87%. In 2021, 1,392 sick dogs were received, with 58 dogs diagnosed with pathogenic E. coli infections, corresponding to an annual infection rate of 4.17%. The two-year average infection rate of 4% indicates a relatively stable trend over the study period (Table 1). Table 1 Infection rate of canine pathogenic E. coli at the Kawakita Animal Hospital from 2020 to 2021 year Number of visits Number of confirmed cases Morbidity rate (%) 2020 1837 71 3.87 2021 1392 58 4.17 All 3229 129 4 3.2.1 Pathogenic E. coli infection in dogs of different sexes As shown in Table 2, males represented 61.24% of canine E. coli infections, which was higher than the 38.76% observed in females, but the difference was not statistically significant (P = 0.529). 3.2.2 Pathogenic E. coli infection in different breeds of dogs 3.2.3 Pathogenic E. coli infection in dogs in different seasons As shown in Table 2, the highest proportion of infections occurred in spring, accounting for 34.11% of the total annual number of infected dogs. Summer infections represented 24.03%, fall infections accounted for 19.38%, and winter infections contributed to 22.48% of the annual total. Statistical analysis revealed no significant difference between the seasonal infection rates (P = 0.819). 3.2.4 The effect of immunization and non-immunization on the infection of dogs with pathogenic E. coli bacteria Among the dogs infected with pathogenic E. coli , 50.39% were immunized, while 49.61% were unimmunized. Statistical analysis indicated no significant difference between the two groups (P = 0.672). 3.2.4 Pathogenic E. coli infections in dogs at different ages As shown in Table 2, 5.43% of the total number of dogs infected with pathogenic E. coli were lactating, 49.61% were juvenile, 38.76% were adult, and 6.20% were elderly. Chi-square analysis (cross-tabulation) revealed a significant association between the age category and E. coli infection (χ² = 9.033, P = 0.029, P < 0.05). Infection rates were significantly higher in suckling pup than in the adult (P = 0.012) and senior (P = 0.007) age groups. All showed significant differences indicating that suckling pup were more susceptible to infection. These results suggest that the prevalence of E. coli infection varies across different age groups. Specifically, the proportion of puppies infected with pathogenic E. coli (49.61%) was significantly higher than the proportion of adult dogs infected with E. coli (38.76%) and the proportion of older dogs (6.2%)(P = 0.029). Table 2 The results of χ² test for the impact of various factors on the incidence rate of pathogenic E. coli in dogs Characteristics Total Infected Uninfected P χ 2 (n=3100) 129(4.1%) 3229(95.9%) Sex Male vs. female 1891(58.56) vs. 1338(41.44) 79(61.24) vs. 50(38.76) 1812(58.45) vs. 1288(41.55) 0.529 0.397 Period 0.819 0.928 Spring vs. Summer 991(30.69) vs. 798(24.71) 44(34.11) vs. 31(24.03) 947(30.55) vs. 767(24.74) 0.560 0.339 Spring vs. Autumn 991(30.69) vs. 707(21.90) 44(34.11) vs. 25(19.38) 947(30.55) vs. 682(22.00) 0.352 0.865 Spring vs. Winter 991(30.69) vs. 733(22.70) 44(34.11) vs. 29(22.48) 947(30.55) vs. 704(22.71) 0.622 0.243 Summer vs. Autumn 798(24.71) vs. 707(21.90) 31(24.03) vs. 25(19.38) 767(24.74) vs. 682(22.00) 0.721 0.127 Summer vs. Winter 798(24.71) vs. 733(22.70) 31(24.03) vs. 29(22.48) 767(24.74) vs. 704(22.71) 0.942 0.005 Autumn vs. Winter 707(21.90) vs. 733(22.70) 25(19.38) vs. 29(22.48) 682(22.00) vs. 704(22.71) 0.675 0.176 Immunity Unimmunized vs. Immunity 1568(48.56) vs. 65(50.39) vs. 1503(48.48) vs. 0.672 0.18 1661(51.44) 64(49.61) 1597(51.52) Age category 0.029* 9.033 Suckling pup vs. Puppy 77(2.38) vs. 1405(43.51) 7(5.43) vs. 64(49.61) 70(2.26) vs. 1341(43.26) 0.070 3.293 Suckling pup vs. Adult 77(2.38) vs. 1433(44.38) 7(5.43) vs. 50(38.76) 70(2.26) vs. 1383(44.61) 0.012* 6.313 Suckling pup vs. Elderly 77(2.38) vs. 314(9.72) 7(5.43) vs. 8(6.20) 70(2.26) vs. 306(9.87) 0.007* 7.716 Puppy vs. Adult 1405(43.51) vs. 1433(44.38) 64(49.61) vs. 50(38.76) 1341(43.26) vs. 1383(44.61) 0.148 2.091 Puppy vs. Elderly 1405(43.51) vs. 314(9.72) 64(49.61) vs. 8(6.20) 1341(43.26) vs. 306(9.87) 0.108 2.577 Adult vs. Elderly 1433(44.38) vs. 314(9.72) 50(38.76) vs. 8(6.20) 1383(44.61) vs. 306(9.87) 0.399 0.711 Note: p<0.05 labeled "*" indicates Significant correlation between variables 3.2.5 The primary pathogenic factors of E. coli. As shown in Supplemental Figure 1, we integrated the data and utilized a Scale-Location plot to assess potential influential factors contributing to the elevated pathogenic rate of E. coli. Our objective was to identify whether specific factors, such as breed (e.g., Poodles), age group (e.g., puppies), or seasonal variations (e.g., spring), are influencing the pathogenicity of E. coli . The standardized residuals for Poodles, puppies, and the spring season were 48, 43, and 13, respectively, which are notably higher compared to other factors. In addition, factors such as male gender and vaccination status showed slightly elevated residual values, suggesting that these elements also contribute to the primary influences on the pathogenic rate of E. coli . 3.3 Morphological observation of pathogenic E. coli 3.3.1 bacterial isolation and culture The diluted samples were evenly spread on prepared MacConkey agar plates and incubated at 37℃ for 24 h. As shown in Figure 3-A, distinct colonies of varying sizes with flattened shapes, smooth surfaces, and neat edges were observed. The colonies appeared red and moist. Subsequently, a well-developed colony was selected using an inoculation loop, subcultured onto a fresh plate, and incubated again at 37°C for 24 hours. As observed in Figure 3-B following dilution and subculturing, colonies grew in the areas where the inoculation loop had passed, with the colony density becoming lower and the colonies displaying a pink coloration. The strain obtained from the culture was subjected to Gram staining and examined under a light microscope. The results revealed that The bacterial cells were observed as short rods with blunt, rounded ends, displaying a Gram-negative staining pattern appearing red under the microscope. The cells exhibited uniform size approximately 1-3 μm in length and 0.5 μm in width, arranged singly or in pairs, with no evidence of spore formation. The absence of spores, combined with the rod-shaped morphology and Gram-negative characteristics, aligns with typical features of E. coli . Additionally, the moist, red colonies on MacConkey agar suggest lactose fermentation capability, further supporting the identification of Enterobacteriaceae members such as E. coli . The Gram-staining morphology of the strain is shown in Figure 3-C and 3-D. 3.4. Results of homology analysis for DNA identification and MLST Gene sequencing was performed on the 27 bacterial isolates obtained from the cultures, and the sequence results were compared against a database using BLAST. The analysis confirmed that all 27 isolates were identified as E. coli . Furthermore, Multi-Locus Sequence Typing (MLST) analysis was conducted, and the results corroborated the identification, confirming that all isolates belonged to E. coli (see Supplemental Data Table 4). 3.4.1 Comparative results of whole gene sequencing GO functional classification annotation databases In groups A-C, cellular processes represented the largest proportion of biological processes, with membrane-associated components being the most abundant in the cellular component category, and catalytic activity being the most prevalent molecular function. According to the COG functional gene annotation classification, cellular processes accounted for 18% of the total, metabolic processes for 14%, and catalytic activity for 11%. The highest predicted general function category was "general function," followed by "carbohydrate transport and metabolism," and "amino acid transport and metabolism." In between-group comparisons, immune system processes were significantly higher in group A compared to groups B and C, likely due to activation in response to infection, allergies, inflammation, or stress. Additionally, "other organism parts" were significantly lower in groups A and F than in the other four groups, possibly due to tissue or organ damage affecting specific functions, such as organ failure, dysfunction, nutritional deficiencies, or aging. These differences in functional profiles between groups A and B-C suggest that the pathogenic E. coli strains from Shunqing District (group A) had a more significant impact on tissue and organ functions in dogs than those from Gaoping District (group B) or Jialing District (group C) (see Supplemental Data Figure 2). 3.4.2 Comparative results of whole gene sequencing virulence factor annotation databases As shown in Supplemental Data Table 5, whole genome sequencing of pathogenic E. coli strains and subsequent BLAST database comparison revealed the presence of multiple virulence factors in all six strains. Among these, the most common virulence factors included fimbriae and lipopolysaccharide (LPS). Strains numbered 1-6 and 12-9 exhibited virulence factors related to Brk, while strains numbered 12-4 and 12-1 displayed Ptx. Additionally, Pseudomonas aeruginosa PAO1 was identified as a major shared virulence factor across groups A-C, including lipopolysaccharides, secreted toxins, elastases, histolytic enzymes, and oxidative stress response factors. Burkholderia pseudomallei K96243 and Aeromonas hydrophila ATCC 7966 were also identified as shared sources of multiple virulence genes, with Aeromonas hydrophila having the highest number of virulence genes among all groups. When comparing different groups, the number of virulence genes, including those from Pseudomonas aeruginosa PAO1, was significantly higher in group A compared to groups B and C, suggesting that the E. coli strain from Nanchong (group A) exhibited virulence resulting from mixed infections with various virulence genes. 3.4.3 Comparison of whole gene sequencing proximate strain databases As shown in Supplemental Data Figure 3, BLAST comparison of the whole gene sequences of 6 E. coli strains revealed a close resemblance to E. col i K-12, E. coli SE11, E. coli SE15, and E. coli 11128. Groups A-C E. coli strains carried a total of 1614 resistance genes (the major drug resistance genes As shown in Supplemental Data Table 6), primarily associated with antibiotic efflux, alteration of antibiotic targets, and antibiotic target protection. The detection of increased antibiotic efflux expression suggests that antibiotic therapy may be less effective, necessitating alternative therapeutic strategies to combat resistance. The study and monitoring of antibiotic resistance mechanisms in these bacteria are crucial for the development of new antibiotics and for addressing antibiotic-resistant infections. 3.4.4 Drug sensitivity test results The drug sensitivity test was conducted on canine pathogenic E. coli strains, and the results fell within the acceptable range when compared to established standards for measuring the inhibition zones of various antibiotics. As shown in the figure below, the pathogenic E. coli strains exhibited a decreasing concentration around the drug sensitivity discs on the agar plate, indicating sensitivity to the drugs. The lack of uniform diffusion in all directions further suggested some degree of resistance to certain antibiotics. The size of the diffusion zone was directly related to both the concentration of the antibiotic discs and the bacterial density. Five strains of E. coli were randomly tested for resistance to 17 different antibiotics. The results indicated that canine pathogenic E. coli exhibited varying degrees of resistance to different drugs, as summarized in (supplemental data Table 7). Notably, the strains showed 100% sensitivity to ampicillin, cefazolin, cefoxitin, ceftriaxone, gentamicin, ciprofloxacin, levofloxacin, norfloxacin, doxycycline, chloramphenicol, and others, with moderate resistance observed for some strains to amoxicillin, streptomycin, and butyl carbamate. The varying patterns of resistance suggest that the local frequency and dosage of these antibiotics might influence the observed resistance, confirming the clinical effectiveness of certain drugs (Supplemental Data Figure 4). 4. Discussion In this study, we found that the infection rate of canine pathogenic E. coli disease in the Nanchong area was 3.87% in 2020 and increased to 4.17% in 2021, with an average annual infection rate of 4%. This is a significant increase compared to the 1.6% pathogenic E. coli infection rate in the UK, which may be related to the environment or rearing environment [37]. The higher infection of pathogenic E. coli in the Nanchong area compared to the United Kingdom could be attributed to differences in breeding environments and insufficient attention to the diet of pet dogs by their owners. Additionally, the abundance of animals in Nanchong and the multiple transmission pathways of pathogenic E. coli contribute to the frequent occurrence of the disease. This elevated prevalence of antimicrobial-resistant E. coli in domestic dogs may be further contextualized by environmental and behavioral risk factors identified in Canadian settings. A cross-sectional study analyzing dogs frequenting parks in South-Western Ontario found that 23.5% of dogs harbored antimicrobial-resistant E. coli , with resistance most prevalent against ampicillin (68%), tetracycline (42%), and trimethoprim-sulfamethoxazole (19%) Notably, dogs visiting parks more than twice weekly had 1.8-fold higher odds of carrying resistant strains compared to infrequent visitors, suggesting environmental transmission through fecal-oral routes or direct contact in shared spaces[38]. Although the relative infection rate was stable, there is still a risk of transmission. Significant sex differences were observed in pathogenic E. coli infections, with males exhibiting higher infection rates than females. Teddy, Corgi, and Pomeranian are the commonly infected breeds, and the infection rate of small dogs is higher. Moreover, dogs in the Nanchong area are at risk of infection with pathogenic E. coli throughout the year, especially in spring and summer; warmer temperatures and increased humidity may be the main causes of the high infection. In this study, the immunization status has little effect on the incidence of E. coli disease, but immunization remains an important means of preventing infection[39]; the immunized dogs are better able to fight the pathogen and have lower morbidity. In addition, significant differences were found in infection with pathogenic E. coli in dogs of different ages. suckling pup and puppies have higher infection rates than adult and older dogs, which may be related to the weaker immunity of puppies. Other studies also found that the younger the animal, the more susceptible to pathogenic E. coli infection [40]. After follow-up comparative investigation, we found that the living conditions of dogs in Jialing and Gaoping districts are better, and the incidence of pathogenic E. coli is lower, but the living conditions in Nanbu and Yingshan counties are worse, and the incidence of the disease is relatively higher.Subsequently, we conducted follow-up visits and investigations on dogs living in similar environments and found that living conditions, food, water sources, and the frequency of contact with other animals are the main factors contributing to the infection of pathogenic E. coli in dogs. When owners restrict their dogs' activity areas to places with less animal waste and lighter soil contamination, the infection rate shows a slight decrease. This indicates that fecal matter and soil pollution are also potential influencing factors. The incidence of pathogenic E. coli infection in dogs in the Nanchong area is relatively low but still requires attention. Dogs may be susceptible to infection in different seasons and ages, especially in spring and puppies. Symptomatic treatment and the use of antibiotics are key in the treatment process [41]. To effectively prevent and control the occurrence of canine pathogenic E. coli disease, owners should strengthen the management of environmental hygiene [42], boost puppy and suckling pup immunity, monitor pet health, and promptly isolate and treat E. coli -infected dogs to prevent bacterial spread. When medication was used, it is important to avoid frequent or excessive reliance on antibiotics. The pathogenic rate of E. coli can be reduced by improving living conditions, reducing contact with unfamiliar animals, and avoiding the consumption of contaminated food and water. For populations with high animal density, it is recommended to vaccinate dogs against pathogenic E. coli .Veterinarians and public health departments should pay closer attention to the use of antibiotics. Compared to the types of antibiotics previously used, there should be an increased focus on introducing or developing new antibiotics or designing new treatment protocols specifically for pathogenic E. coli . Additionally, it is important to keep statistics on the frequency of use of various types of antibiotics and the diseases they are used to treat, and to develop strategies to counteract strains similar to tetracycline-resistant bacteria [43]. Many owners in the rearing process, ignore bacterial resistance and antibiotic use, leading to antibiotic abuse, which is the main cause of bacterial resistance [44]. China's intensive livestock farming has enhanced pathogenic E. coli adaptability, promoting diversification of virulence factors (e.g., adhesins, toxins) across regions and over time. Analyzing these factors is key to developing targeted interventions against outbreaks.In this study, 27 strains of pathogenic E. coli were obtained through bacterial isolation and culture and 16rs DNA PCR identification from canine faces in the Nanchong area. Six strains were selected for whole gene sequencing. The sequencing results were compared by using blast to analyze the annotation of virulence factors and drug resistance function of pathogenic E. coli in the Nanchong area, in which the most important virulence factors were Fimbriae , LPS , Brk , Ptx, Pertactin [45]. also known as E. coli bacterial pili. LPS act as virulence factors, while high-dose LPS disrupt endothelial barrier function [46]. In a mouse model, BrkA mutants showed attenuated respiratory tract infectivity compared to wild-type strains. These mutants also exhibited increased complement sensitivity in vitro, confirming BrkA's role in resisting complement-mediated killing. Moreover, multiple DeltabrkA mutants derived from Tohama I in mice exhibited severe defects within the first week after inoculation. This defect was present even in complement-deficient mice, revealing a complement-independent phenotype of BrkA in respiratory tract infections[47] PTX , utilizing its unique ADP-ribosylation capability, disrupts and perturbs the intricate signaling processes within cells. This interference impedes the normal functioning of cells, leading to dysfunction and potentially triggering cell death. In essence, PTX impairs host cell signaling [48], while pertactin mediates B. pertussis epithelial adhesion and immune escape via expression plasticity, sustaining infection [49]. The isolates of pathogenic E. coli had certain drug resistance, mainly expressed as antibiotic efficacy. All the strains have some sensitivity to antibiotics through drug sensitivity tests; tetracycline sensitivity is the lowest, at 60%, followed by amoxicillin, streptomycin, and butamocarbamol, with a sensitivity of 80%. Most of the E. coli colonization factors are expressed as adhesins. A study conducted on canine E. coli isolates from clinical samples in the northeastern U.S. revealed resistance rates ranging from 0.4% (amikacin) to 34.3% (ampicillin). Survey data indicated significant increasing trends in resistance to several drugs, including cephalosporins, enrofloxacin, and tetracycline. Similarly, Cummings' study highlighted widespread AMR and MDR in pathogenic E. coli from both dogs and cats, largely attributed to extensive antimicrobial use in veterinary practice. High resistance rates were observed against β-lactams, fluoroquinolones, and sulfonamides, with notable regional variations in the resistance profiles[50]. On E. coli isolates from clinical canine samples in New York, USA, revealed a significant increase in MDR among the bacterial strains due to the extensive use of antibiotics and other medications. Specifically, MDR was prevalent, with high resistance rates to ampicillin and cephalosporins, showing increasing trends over time. Resistance to gentamicin and enrofloxacin also rose progressively, while sulfonamides exhibited persistently high resistance [51]. Therefore, it can be seen that the antibiotic resistance of pathogenic E. coli infections in dogs varies from region to region. Antibiotic efflux pumps are key in bacterial resistance, expelling antibiotics from cells to lower their intracellular concentration and allow bacterial survival. Antibiotic efflux pumps are major contributors to enhanced bacterial resistance by expelling antibiotics from cells to reduce drug concentration, allowing bacteria to combat antibiotics [52]. Mutations in bacterial genomes can alter antibiotic target proteins, reducing drug binding affinity or disrupting their function, thereby conferring resistance. This adaptive evolution is a key driver of resistance development. Furthermore, mobile genetic elements (e.g., plasmids, transposons) enable cross-species dissemination of resistance genes through conjugation or transposition, accelerating resistance spread. These vectors act as "resistance propagators," rapidly dispersing resistance traits across bacterial populations[53]. Notably, Bacterial biofilms act as protective barriers, impeding antibiotic penetration. Within biofilms, bacteria exhibit reduced metabolic activity and antibiotic susceptibility. The biofilm matrix (e.g., polysaccharides, proteins) further neutralizes antimicrobial agents. Of public health concern, pathogenic E. coli resistance genes can transfer via mobile genetic elements to Salmonella or commensal strains, accelerating resistance dissemination under antimicrobial pressure [54]. Consequently, biofilm formation represents another pivotal strategy bacteria employ to withstand antibiotic assault. In conclusion, antibiotic efflux pumps, coupled with bacterial genomic mutations, interbacterial genetic exchange, and biofilm formation, constitute a complex network underpinning bacterial resistance. Understanding these mechanisms is paramount for developing novel antibiotics, optimizing existing treatment protocols, and formulating effective resistance prevention and control strategies. The rampant abuse of antibiotics in China has become a pressing issue, significantly exacerbated the spread of bacterial resistance, and compromise the efficacy of antibiotics as a "lifesaving shield," rendering numerous once-manageable infectious diseases more complex and difficult to treat. To effectively combat health threats like canine colibacillosis, it's crucial to regulate antibiotic use in pet hospitals for precise medication. Rational antibiotic use and reducing unnecessary prescriptions are key to curbing antibiotic resistance. Concurrently, the urgent need for novel antibiotic development is paramount in the face of growing resistance challenges. These new drugs must possess novel targets and mechanisms of action, capable of penetrating existing resistance barriers and paving the way for innovative treatment strategies against resistant pathogens. Furthermore, we must actively explore and promote alternative therapies such as immunotherapy and bacteriophage therapy, which not only diversify treatment options for resistant infections but also alleviate the pressure on antibiotic usage, further hindering the progression of resistance. We must adopt a multifaceted approach to tackle the challenge of antibiotic misuse by strengthening regulation, promoting rational use, developing new drugs, advocating alternative therapies, and enhancing monitoring and prevention, thus safeguarding the health of humans and animals and ensuring the sustainable use of antibiotics in medical treatment. 5. Conclusion In conclusion, canine pathogenic E. coli disease has a large negative impact on the overall health status of dogs and social security and health. This study investigates the epidemiological pathogenicity of domestic dogs in the Nanchong area and isolation of pathogenic E. coli to analyze the whole gene, focusing on the virulence factor and its resistance to complete the in-depth analysis, the preliminary confirmation of the current status of the rate of infection pathogenic E. coli and its strains of pathogenicity. The epidemiological status of pathogenic E. coli was shown in Figure 4. Declarations Acknowledgements We would like to thank Chuanbei Animal Hospital in Nanchong City for their support of this research. In addition, we would like to acknowledge all the animal hospitals and pet owners who allowed the use of samples in this study. Authors ’ contributions Bangyuan Wu: Conceptualization, Formal analysis, Data curation, Validation. Kai Li: Formal analysis, Writing-original draft, Data curation. Aifei Du: Formal analysis, Data curation. Shunjie Tang: Conceptualization, Formal analysis. Shaohua Feng: Conceptualization, Supervision. Shibin Yuan: Supervision, Conceptualization, Methodology, Writing-Review & Editing. All authors read and approved the final manuscript. Funding This study was fully supported by the Nanchong Key Laboratory of Wildlife Nutrition Ecology and Disease Control, Sichuan, China (NCKL202201), the National Natural Science Foundation of China (32370557), Key Project of the Joint Fund for Science and Education of Sichuan Province(2024NSFSC1967) and the Fundamental Research Funds of China West Normal University (Project No. 20A003). Data availability Data is provided within the manuscript or supplementary information files. Ethics approval and consent to participate Ethical approval the study was conducted in accordance with the guidelines established by the International Animal Ethics Committee, and the treatment of the animals adhered to the guidelines set forth by the Animal Care Committee of China West Normal University (2024LLSC0052). 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Supplementary Files SupplementalDataTable.docx RNAsequences.xlsx SupplementalDataFigure.docx DNAsequences.xlsx Proteinsequences.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":228042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClassification of Canine Diseases in Nanchong from January 2020 to December 2021\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/2e61338b17bca0815f8934e1.png"},{"id":95529630,"identity":"37659a2c-89d8-499f-9a5f-39b2dd111348","added_by":"auto","created_at":"2025-11-10 10:17:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":241745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatistics on the infection rate of pathogenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in different breeds of dogs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/876580741331d0742474d261.png"},{"id":95528722,"identity":"3bb1ab7c-7596-4620-b2b4-8b36f87272b3","added_by":"auto","created_at":"2025-11-10 10:16:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":799780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial isolation culture morphology as well as microscopy morphology observation results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B): morphology of bacterial isolation and culture; (C-D): the morphology of strain microscopy.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/5368f553cab54d3f9f141165.png"},{"id":95503631,"identity":"a1269d64-3f43-44a1-b443-4e6554430784","added_by":"auto","created_at":"2025-11-10 05:40:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":156468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCurrent status of Nanchong pathogenic E. coli in dogs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/64d6c3d98965d019470b81be.png"},{"id":95800789,"identity":"b7f972c2-03ab-4251-9615-299636d41d27","added_by":"auto","created_at":"2025-11-13 08:23:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2502286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/f3968ef5-0ce4-40b3-b65b-371d08c641a7.pdf"},{"id":95528905,"identity":"36cf6cad-593b-49ff-b87d-3e74d5d5d0ab","added_by":"auto","created_at":"2025-11-10 10:16:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1286413,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/5ace92bdad65a8fcaddb1281.docx"},{"id":95503628,"identity":"27adab8d-e97b-4b5a-92bb-c5434ef21b98","added_by":"auto","created_at":"2025-11-10 05:40:34","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29849,"visible":true,"origin":"","legend":"","description":"","filename":"RNAsequences.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/179d14ff95ed5f106363b8d3.xlsx"},{"id":95503637,"identity":"1581fd80-5d1a-47e2-832a-d1171901c0ab","added_by":"auto","created_at":"2025-11-10 05:40:34","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1267563,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/99e8440d1809e5a7261caec9.docx"},{"id":95503639,"identity":"ccf6f594-b8ca-4c35-89e0-ebc13ccbda18","added_by":"auto","created_at":"2025-11-10 05:40:34","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":79644,"visible":true,"origin":"","legend":"","description":"","filename":"DNAsequences.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/206c7254fc1e65e7d392ba4c.xlsx"},{"id":95503642,"identity":"a1b396d2-953f-4408-8620-9d297ce00e81","added_by":"auto","created_at":"2025-11-10 05:40:34","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":197967,"visible":true,"origin":"","legend":"","description":"","filename":"Proteinsequences.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7708388/v1/b0489933415e111a6c09dda3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epidemiologic investigation of pathogenic Escherichia coli in domestic dogs in Nanchong area","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePathogenic \u003cem\u003eE. coli\u003c/em\u003e is a significant safety hazard in different areas of pig, chicken, and duck farming[1], and its features, including difficult to cure, high mortality rate, and very easy to recur, make it a persistent challenge in livestock production [2]. Animals infected with pathogenic \u003cem\u003eE. coli\u003c/em\u003e will cause different body inflammation [3], such as urethritis [4], gastroenteritis [5], umbilical cord infection and vitellicle infection [6]. It is essential to consider the antimicrobial resistance (AMR) and concurrent infections when treating \u003cem\u003eE. coli\u003c/em\u003e. In addition, the endotoxin released by pathogenic\u003cem\u003e\u0026nbsp;E. coli\u0026nbsp;\u003c/em\u003eshould also pay attention to [7], which is highly toxic and can cause fever [8], sepsis, toxemia [9], pericarditis [10], nephritis [11], and even induce to death [12]. The initial symptoms of canine\u003cem\u003e\u0026nbsp;E. coli\u0026nbsp;\u003c/em\u003edisease are not obvious [13], and it is not easy to cause the attention of dog owners. In the late stage, the sick dog will present depressed, loss of appetite or complete abolition, the severe dehydration phenomenon, poor skin elasticity, eye sockets sunken, limb weakness or body temperature drop [14], and will quickly occur diarrhea, discharge viscous yellowish-white, yellowish-green, or greenish dilute feces, emitting an unpleasant Fishy odor [15]. Pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e infections in puppies often result in diarrhea that produces air bubbles and food residues due to lack of digestion, causing secondary fecal contamination of body surfaces (tail, hind legs, anus).\u003c/p\u003e\n\u003cp\u003ePathogenic \u003cem\u003eE. coli\u003c/em\u003e invades the host via the oral or nasal cavity, evades antimicrobial defenses, and proliferates in the small intestine. It colonizes the intestinal mucosa through the bacterium\u0026apos;s colonization factor (pilus or adhesins) [16], which binds to the cells\u0026apos; specific receptors on the mucous membrane\u0026apos;s surface, causing inflammation. Pathogenic \u003cem\u003eE. coli\u003c/em\u003e can produce endotoxin [17], enterotoxin\u0026nbsp;[18], and colistin\u0026nbsp;[19]. When pathogenic \u003cem\u003eE. coli\u003c/em\u003e disintegrates, it releases lipopolysaccharides, and the lipid A component can cause endotoxemia\u0026nbsp;[20]. Enteroproducing virulent \u003cem\u003eE. coli\u003c/em\u003e can infect epithelial cells of the small intestine, colonize and proliferate on the cells, and produce enterotoxins\u0026nbsp;[21]. \u003cem\u003eLT\u003c/em\u003e and \u003cem\u003eST\u0026nbsp;\u003c/em\u003eas two important enterotoxins; \u003cem\u003eLT\u0026nbsp;\u003c/em\u003e(Heat-Labile Enterotoxin) activates adenylyl cyclase in intestinal epithelial cells, increasing cAMP production, which causes hypersecretion, diarrhea, and dehydration[22]. \u003cem\u003eST\u003c/em\u003e (Heat-Stable Enterotoxin) activates guanylyl cyclase (cGMP) in ileal epithelial cells, causing secretory diarrhea; many \u003cem\u003eE. coli\u003c/em\u003e produce colicin V (ColV) plasmids[23, 24], This is linked to the bacterium\u0026rsquo;s ability to cause sepsis. Additionally, specific toxin-producing\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e (ETEC) can invade and damage intestinal mucosa cells, with invasiveness related to the presence of a plasmid\u0026nbsp;[25].\u003c/p\u003e\n\u003cp\u003eRuminants such as sheep, deer, and cattle are the primary hosts of\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e [26], and the range of the animals and complex environmental influences can cause \u003cem\u003eE. coli\u003c/em\u003e migration. In the study of the mechanism of\u003cem\u003e\u0026nbsp;E. col\u003c/em\u003ei invasion of soil and its interactions with indigenous microorganisms, it has been found that pathogenic \u003cem\u003eE. coli\u003c/em\u003e forms a complex migratory chain with the environment [27], thus threatening the development of the farming industry and even to the health of human beings. A mount of\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e exists in the feces of infected animals, and according to the survival characteristics of\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e [28], it can survive for a long time in the adapted environment. The feces of animals infected with pathogenic \u003cem\u003eE. col\u003c/em\u003ei migrate through the irrigation of farmland, flies, birds, rainfall flushing, and groundwater flow, forming a closed-loop contamination of food [29] and water [30], which threatens the safety of public health. Therefore, managing the scope of activities of feeding animals is very important for preventing and controlling pathogenic \u003cem\u003eE. coli\u003c/em\u003e infection. Moreover, Pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e is a zoonotic disease [31]; the transmission is mainly through food, water, and intimate contact [32], of which food transmission is the primary way. However, due to the simple transmission pathway,\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e is always a threat to public health safety and the development of animal husbandry. Though the pathogenic \u003cem\u003eE. coli\u003c/em\u003e infection situation has improved, there is still a risk of infection outbreaks, especially in dog farms and pet dog breeding [33].\u003c/p\u003e\n\u003cp\u003eIn northeast China, the infection of pathogenic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003einfections in dogs is as high as 63.75% [34], a statistic that reveals a serious challenge to canine health in the region. In the veterinary and public health fields, high infection rates can lead to a decline in the health of canine populations [35], affecting their quality of life and productivity and highlighting the potential risk of transmission of zoonotic diseases to human health, such as urinary tract infections, nephritis [36].\u003c/p\u003e\n\u003cp\u003eNanchong, the second most populous city in Sichuan China, has more families of pet dogs, farm dogs, and stray dogs, and a wide range of activities of these dogs will produce secondary pollution. Hence, the infection rate has been high for a long time. In the present study, the infected with pathogenic \u003cem\u003eE. coli\u003c/em\u003e dogs were analyzed through the different seasons, different breeds of dogs, different genders, different age incidence in the Nanchong region, the collection of feces from typical diseased dogs, and the isolation of identified Pathogenic \u003cem\u003eE. coli\u003c/em\u003e was collected, and the isolation and identification of pathogenic \u003cem\u003eE. coli\u003c/em\u003e, drug sensitivity test, and genotyping were carried out to obtain the corresponding rules, which can be used as the reference data for the prevention of pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e infections in animal husbandry, prevention and control of the northeastern region of Sichuan. This study establishes Nanchong as a sentinel surveillance site to reduce pathogenic \u003cem\u003eE. coli\u003c/em\u003e infection prevalence in canine populations through molecular epidemiological characterization of virulence determinants and antimicrobial resistance patterns, thereby developing targeted biosecurity interventions for effective zoonotic transmission control.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cem\u003e2.1 Sample and data collection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe case data for this study were obtained from The Kawakita Animal Hospital in Nanchong City. The hospital was established in 2013, a specialized and well-equipped veterinary institution for dogs and cats, approved by the Nanchong Municipal Animal Husbandry and Veterinary Medicine Department. It focuses on the diagnosis and treatment of pet diseases. Data collection was conducted from January 2020 to December 2021. During this period, a total of 3,229 canine cases were diagnosed, with more than 600 owners contacted via randomized telephone follow-up. The consultation area covered various districts within Nanchong City, including Shunqing District, Xichong County,Jialing District, Gaoping District, Peng\u0026rsquo;an County, Yingshan County, and Langzhong City, as well as neighboring cities such as Dazhou, Suining, and Chengdu.\u003c/p\u003e\n\u003cp\u003eSamples were grouped by geographic region: Group A (Shunqing District), Group B (Gaoping District), and Group C (Jialing District). Based on the statistical distribution of pathogenic \u003cem\u003eE. coli\u003c/em\u003e cases in Nanchong, representative cases were selected from each group for sampling to ensure geographical representation.\u003c/p\u003e\n\u003cp\u003eTo account for seasonal variation, in Nanchong City, a follow-up survey of diseased dogs was conducted throughout the four seasons across various districts and counties(spring, summer, autumn, and winter) If a sampled dog recovers, the next eligible dog within the vicinity is selected to maintain full coverage of Nanchong\u0026rsquo;s districts and counties. Each dog was continuously observed until fecal discharge. Feces were collected aseptically into sterile containers, with the outer layer discarded using sterile swabs. The inner fecal layer was placed into cryotubes, stored in a portable dry ice refrigerator, and transferred to the laboratory for preservation at -80℃ (Yuan et al., 2024). Specifically, 13 samples were collected from Shunqing District, 7 from Xichong County, 7 from Gaoping District, and 3 from Jialing District.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2\u0026nbsp;\u003c/em\u003e\u003cem\u003eIsolation, Genotyping, and Drug Sensitivity Testing of Pathogenic E. coli\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIsolation and identification of strains are suspected pathogenic \u003cem\u003eE. coli\u003c/em\u003e strains were isolated from fecal samples through enrichment culture, followed by purification using repeated streaking, and stored at -80℃. Samples were processed by Shanghai Biotechnology Company for strain identification and full-genome sequencing. This allowed for analysis of phylogenetic relationships, annotation of virulence factors, and assessment of antibiotic resistance genes.\u003c/p\u003e\n\u003cp\u003eBacterial culture and purification strain proliferation culture: Luria-Bertani (LB) broth was prepared using yeast extract (Beijing Auboxing Biotechnology Co., Ltd.) and tryptone (Qingdao HaiBo Biotechnology Co., Ltd.). Approximately 1-2 g of fecal sample was inoculated and incubated at 37℃ for 24 h. Purification culture: MacConkey agar (Qingdao HaiBo Biotechnology Co., Ltd.) was used for isolation. Serial dilutions (10^1 to 10^5) of the samples were plated using a sterile spreader. Plates were incubated at 37℃ for 24 h. Single colonies were repeatedly purified for three generations. Strain Preservation: Purified colonies were mixed with 40% sterilized glycerol and stored in EP tubes at -20℃ (Ribeiro-Almeida et al., 2024).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3 Genome Extraction, PCR Amplification, and Sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA extraction: DNA was extracted using the BigDye Sequencing Reaction Kit (SK1201-UNIQ-10) following manufacturer instructions. PCR amplification: PCR was performed using the BigDye Sequencing Reaction Kit (4337455, Shanghai Sangon). The thermal cycle was as follows: initial denaturation at 94℃ for 5 min, followed by 35 cycles of 94℃ for 30 s, 55℃ for 35 s, and 72℃ for 1 min, with a final extension at 72℃ for 8 min.\u003c/p\u003e\n\u003cp\u003eGel electrophoresis and purification: PCR products were analyzed via agarose gel electrophoresis. Desired DNA bands were excised and purified according to the instructions of SK1131. Sequencing primers: M13-47 primer (AGGGTTTTCCCAGTCACG) was used for sequencing (Supplemental Data Table 1). Sequencing reaction and processing: PCR sequencing was performed in PCR tubes placed on ice. After DNA precipitation with sodium acetate and ethanol, centrifugation was conducted, followed by washing with 70% ethanol. DNA was dried under vacuum, resuspended in TSR buffer, heat-denatured, and cooled on ice before sequencing (supplemental data Table 2).\u003c/p\u003e\n\u003cp\u003eCapillary electrophoresis was conducted with correct positioning and automatic gel loading. Pre-electrophoresis was done at 1.2 kV for 5 min, followed by electrophoresis at 7.5 kV for 2 h. Sequence analysis was performed automatically.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4 Annotation and Phylogenetic Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eVFDB (Virulence Factor Database): The protein sequences of predicted genes were aligned using BLAST against the VFDB, which contains 32,638 genes related to virulence factors across 2,611 entries, all supported by experimental evidence.\u003c/p\u003e\n\u003cp\u003eCARD (Comprehensive Antibiotic Resistance Database): This database contains 2,359 sequences and 3,567 Antibiotic Resistance Ontology (ARO) terms. Sequences were aligned using BLAST to identify resistance-related annotations.\u003c/p\u003e\n\u003cp\u003e16S rRNA gene analysis: Highly conserved and present in all bacteria, the 16S rRNA gene sequences were compared with NCBI\u0026rsquo;s 16S database using BLAST. The top 30 hits were selected for phylogenetic analysis. These sequences were then aligned and used to construct a phylogenetic tree.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5\u003c/em\u003e \u003cem\u003eDrug Sensitivity Test Methods\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe drug sensitivity of pathogenic \u003cem\u003eE. coli\u003c/em\u003e was tested using the Disk Diffusion Test (supplemental data Table 3). MH medium (028050, MH agar Hangzhou Binhe Microbiology Reagent Co., Ltd.) was inoculated with the bacteria, and a sterile cotton swab was used to coat the medium uniformly. Sensitive paper sheets (921171, detection of drug sensitivity tablets, Changde Beekman Biotechnology Co., Ltd.) were placed on the plate, ensuring a center distance of at least 24 mm. After incubating at 37℃\u0026nbsp;for 24 h, the inhibition circles were measured and classified as resistant (R), moderately sensitive (I), or sensitive (S) based on their size. A larger circle indicated greater sensitivity and less resistance, while a smaller or absent circle indicated resistance.\u003c/p\u003e\n\u003cp\u003eInterpretation criteria for the diameter of the circle of inhibition of commonly used drug sensitizers for Enterobacteriaceae.\u003c/p\u003e\n\u003cp\u003eThe diameter of the circle of inhibition reflects the antimicrobial activity of different antibiotics against bacteria at specific concentrations. The experiment tested the antimicrobial activity of 17 antibiotics most commonly used in clinical practice, such as ampicillin, amoxicillin, and cefazolin, against bacteria at specific concentrations, which were categorized as resistant (\u0026le;13 mm), moderately sensitive (14-20 mm), and sensitive (\u0026ge;21 mm), respectively (Humphries et al., 2018).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5\u003c/em\u003e \u003cem\u003eMethods of statistical analysis of survey data and Data analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEXCEL was used to statistically organize the data to obtain the incidence rate of pathogenic E. coli-infected dogs, and the formula was calculated:\u003c/p\u003e\n\u003cp\u003e①\u0026nbsp;different seasonal incidence rate (%) = the number of different seasonal incidences / the total number of cases of pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e disease clinic \u0026times; 100%\u003c/p\u003e\n\u003cp\u003e②\u0026nbsp;different breeds of dogs morbidity (%) = the number of different breeds of dogs onset / the total number of cases of pathogenic \u003cem\u003eE. coli\u003c/em\u003e disease clinic\u0026nbsp;\u0026times;\u0026nbsp;100%\u003c/p\u003e\n\u003cp\u003e③\u0026nbsp;Incidence rate of dogs of different ages (%) = the number of dogs of different ages with disease / total number of cases of pathogenic \u003cem\u003eE. coli\u003c/em\u003e disease\u0026nbsp;\u0026times;\u0026nbsp;100%\u003c/p\u003e\n\u003cp\u003e④\u0026nbsp;Incidence rate of dogs of different genders (%) = number of dogs of different genders/total number of diagnosed cases of pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e disease\u0026times;100%.\u003c/p\u003e\n\u003cp\u003e⑤\u0026nbsp;Immunized or not (%)=Number of immunized or not/total number of cases of pathogenic \u003cem\u003eE. coli\u003c/em\u003e disease\u0026times;100%.\u003c/p\u003e\n\u003cp\u003eThe canine cases were categorized by age: 1-60 days old for suckling pup, 2 months to 12 months old for young dogs, 13 months old-84 months old for adult dogs, and 84 months old and above for senior dogs.\u003c/p\u003e\n\u003cp\u003eAn ANOVA analysis was conducted on dogs infected with pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e, examining the effects of different seasons, ages, genders, and immunity acquisition or loss status following immunization. This analysis was performed using SPSS 26.0 software. A p-value of \u0026lt; 0.05 determined statistical significance.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003e3.1 Morbidity in consulting dogs\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 1, a total of 3,229 sick dogs were collected from January 2020 to December 2021 at The Kawakita Animal Hospital in Nanchong. The diseases were categorized into specific groups based on their clinical presentation. Among the total number of 3,229 sick dogs, gastrointestinal diseases were the most prevalent, with 733 cases, accounting for 22.7% of the total number of dogs examined. This was followed by skin diseases, which affected 505 dogs (15.64%). Infectious diseases were the third most common, with 384 affected dogs (11.89%), while genitourinary diseases were recorded in 300 dogs, representing 9.29% of the total cases. These results underscore the significance of gastrointestinal and dermatological conditions as the most frequent health issues observed in the population of sick dogs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2. Annual incidence of pathogenic E. coli\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn 2020, a total of 1,837 sick dogs were admitted, among which 71 were diagnosed with pathogenic infections, resulting in an annual infection rate of 3.87%. In 2021, 1,392 sick dogs were received, with 58 dogs diagnosed with pathogenic \u003cem\u003eE. coli\u003c/em\u003e infections, corresponding to an annual infection rate of 4.17%. The two-year average infection rate of 4% indicates a relatively stable trend over the study period (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 Infection rate of canine pathogenic \u003cem\u003eE. coli\u003c/em\u003e at the Kawakita Animal Hospital from 2020 to 2021\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"88%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.1313%;\"\u003e\n \u003cp\u003eyear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.2929%;\"\u003e\n \u003cp\u003eNumber of visits\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.2929%;\"\u003e\n \u003cp\u003eNumber of confirmed cases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.2828%;\"\u003e\n \u003cp\u003eMorbidity rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.1313%;\"\u003e\n \u003cp\u003e2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.2929%;\"\u003e\n \u003cp\u003e1837\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.2929%;\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.2828%;\"\u003e\n \u003cp\u003e3.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.1313%;\"\u003e\n \u003cp\u003e2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.2929%;\"\u003e\n \u003cp\u003e1392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.2929%;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.2828%;\"\u003e\n \u003cp\u003e4.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.1313%;\"\u003e\n \u003cp\u003eAll\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.2929%;\"\u003e\n \u003cp\u003e3229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.2929%;\"\u003e\n \u003cp\u003e129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.2828%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2.1 Pathogenic E. coli infection in dogs of different sexes\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 2, males represented 61.24% of canine \u003cem\u003eE. coli\u003c/em\u003e infections, which was higher than the 38.76% observed in females, but the difference was not statistically significant (P = 0.529).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2.2 Pathogenic E. coli infection in different breeds of dogs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2.3 Pathogenic\u0026nbsp;\u003c/em\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003cem\u003e\u0026nbsp;infection in dogs in different seasons\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 2, the highest proportion of infections occurred in spring, accounting for 34.11% of the total annual number of infected dogs. Summer infections represented 24.03%, fall infections accounted for 19.38%, and winter infections contributed to 22.48% of the annual total. Statistical analysis revealed no significant difference between the seasonal infection rates (P = 0.819).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2.4 The effect of immunization and non-immunization on the infection of dogs with pathogenic\u0026nbsp;\u003c/em\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003cem\u003e\u0026nbsp;bacteria\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAmong the dogs infected with pathogenic \u003cem\u003eE. coli\u003c/em\u003e, 50.39% were immunized, while 49.61% were unimmunized. Statistical analysis indicated no significant difference between the two groups (P = 0.672).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2.4 Pathogenic\u0026nbsp;\u003c/em\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003cem\u003e\u0026nbsp;infections in dogs at different ages\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 2, 5.43% of the total number of dogs infected with pathogenic \u003cem\u003eE. coli\u003c/em\u003e were lactating, 49.61% were juvenile, 38.76% were adult, and 6.20% were elderly. Chi-square analysis (cross-tabulation) revealed a significant association between the age category and \u003cem\u003eE. coli\u003c/em\u003e infection (\u0026chi;\u0026sup2; = 9.033, P = 0.029, P \u0026lt; 0.05). Infection rates were significantly higher in suckling pup than in the adult (P = 0.012) and senior (P = 0.007) age groups. All showed significant differences indicating that suckling pup were more susceptible to infection. These results suggest that the prevalence of \u003cem\u003eE. coli\u003c/em\u003e infection varies across different age groups. Specifically, the proportion of puppies infected with pathogenic E. coli (49.61%) was significantly higher than the proportion of adult dogs infected with E. coli (38.76%) and the proportion of older dogs (6.2%)(P = 0.029).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 \u0026nbsp;The results of \u0026chi;\u0026sup2; test for the impact of various factors on the incidence rate of pathogenic \u003cem\u003eE. coli\u003c/em\u003e in dogs\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003eUninfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e(n=3100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e129(4.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e3229(95.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eMale \u003cem\u003evs.\u003c/em\u003e female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1891(58.56)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e1338(41.44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e79(61.24)\u003cem\u003evs.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e50(38.76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e1812(58.45)\u003cem\u003evs.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1288(41.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.529\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.397\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeriod\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.819\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.928\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eSpring \u003cem\u003evs.\u003c/em\u003e Summer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e991(30.69)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e798(24.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e44(34.11)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e31(24.03)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e947(30.55)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e767(24.74)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.339\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eSpring \u003cem\u003evs.\u003c/em\u003e Autumn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e991(30.69)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e707(21.90)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e44(34.11)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e25(19.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e947(30.55)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e682(22.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.352\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.865\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eSpring \u003cem\u003evs.\u003c/em\u003e Winter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e991(30.69)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e733(22.70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e44(34.11)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e29(22.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e947(30.55)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e704(22.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.622\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.243\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eSummer \u003cem\u003evs.\u003c/em\u003eAutumn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e798(24.71)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e707(21.90)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e31(24.03)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e25(19.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e767(24.74)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e682(22.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.721\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.127\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eSummer \u003cem\u003evs.\u003c/em\u003e Winter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e798(24.71)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e733(22.70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e31(24.03)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e29(22.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e767(24.74)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e704(22.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eAutumn \u003cem\u003evs.\u003c/em\u003e Winter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e707(21.90)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e733(22.70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e25(19.38)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e29(22.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e682(22.00)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e704(22.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.675\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.176\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eImmunity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003eUnimmunized \u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eImmunity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1568(48.56)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e65(50.39)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e1503(48.48)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 62px;\"\u003e\n \u003cp\u003e0.672\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 50px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e1661(51.44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e64(49.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e1597(51.52)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 457px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge category\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.029*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e9.033\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eSuckling pup \u003cem\u003evs.\u003c/em\u003e Puppy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e77(2.38)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e1405(43.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e7(5.43)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e64(49.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e70(2.26)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e1341(43.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e3.293\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eSuckling pup \u003cem\u003evs.\u003c/em\u003e Adult\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e77(2.38)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e1433(44.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e7(5.43)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e50(38.76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e70(2.26)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e1383(44.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.012*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e6.313\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eSuckling pup \u003cem\u003evs.\u003c/em\u003e Elderly\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e77(2.38)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e314(9.72)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e7(5.43)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e8(6.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e70(2.26)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e306(9.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.007*\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e7.716\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003ePuppy \u003cem\u003evs.\u003c/em\u003e Adult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1405(43.51)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e1433(44.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e64(49.61)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e50(38.76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e1341(43.26)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e1383(44.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e2.091\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003ePuppy \u003cem\u003evs.\u003c/em\u003e Elderly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1405(43.51)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e314(9.72)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e64(49.61)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e8(6.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e1341(43.26)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e306(9.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e2.577\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eAdult \u003cem\u003evs.\u003c/em\u003e Elderly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1433(44.38)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e314(9.72)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e50(38.76)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e8(6.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e1383(44.61)\u003cem\u003evs.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e306(9.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e0.711\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: p\u0026lt;0.05 labeled \u0026quot;*\u0026quot; indicates Significant correlation between variables \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.2.5 \u003cem\u003eThe primary pathogenic factors of E. coli.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Supplemental Figure 1, we integrated the data and utilized a Scale-Location plot to assess potential influential factors contributing to the elevated pathogenic rate of \u003cem\u003eE. coli.\u003c/em\u003e Our objective was to identify whether specific factors, such as breed (e.g., Poodles), age group (e.g., puppies), or seasonal variations (e.g., spring), are influencing the pathogenicity of \u003cem\u003eE. coli\u003c/em\u003e. The standardized residuals for Poodles, puppies, and the spring season were 48, 43, and 13, respectively, which are notably higher compared to other factors. In addition, factors such as male gender and vaccination status showed slightly elevated residual values, suggesting that these elements also contribute to the primary influences on the pathogenic rate of \u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3 Morphological observation of pathogenic E. coli\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3.1 bacterial isolation and culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe diluted samples were evenly spread on prepared MacConkey agar plates and incubated at 37℃ for 24 h. As shown in Figure 3-A, distinct colonies of varying sizes with flattened shapes, smooth surfaces, and neat edges were observed. The colonies appeared red and moist. Subsequently, a well-developed colony was selected using an inoculation loop, subcultured onto a fresh plate, and incubated again at 37\u0026deg;C for 24 hours. As observed in Figure 3-B following dilution and subculturing, colonies grew in the areas where the inoculation loop had passed, with the colony density becoming lower and the colonies displaying a pink coloration. The strain obtained from the culture was subjected to Gram staining and examined under a light microscope. The results revealed that The bacterial cells were observed as short rods with blunt, rounded ends, displaying a Gram-negative staining pattern appearing red under the microscope. The cells exhibited uniform size approximately 1-3 \u0026mu;m in length and 0.5 \u0026mu;m in width, arranged singly or in pairs, with no evidence of spore formation. The absence of spores, combined with the rod-shaped morphology and Gram-negative characteristics, aligns with typical features of \u003cem\u003eE. coli\u003c/em\u003e. Additionally, the moist, red colonies on MacConkey agar suggest lactose fermentation capability, further supporting the identification of Enterobacteriaceae members such as \u003cem\u003eE. coli\u003c/em\u003e. The Gram-staining morphology of the strain is shown in Figure 3-C and 3-D.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4. Results of homology analysis for DNA identification and MLST\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGene sequencing was performed on the 27 bacterial isolates obtained from the cultures, and the sequence results were compared against a database using BLAST. The analysis confirmed that all 27 isolates were identified as \u003cem\u003eE. coli\u003c/em\u003e. Furthermore, Multi-Locus Sequence Typing (MLST) analysis was conducted, and the results corroborated the identification, confirming that all isolates belonged to \u003cem\u003eE. coli\u003c/em\u003e (see Supplemental Data Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4.1 Comparative results of whole gene sequencing GO functional classification annotation databases\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn groups A-C, cellular processes represented the largest proportion of biological processes, with membrane-associated components being the most abundant in the cellular component category, and catalytic activity being the most prevalent molecular function. According to the COG functional gene annotation classification, cellular processes accounted for 18% of the total, metabolic processes for 14%, and catalytic activity for 11%. The highest predicted general function category was \u0026quot;general function,\u0026quot; followed by \u0026quot;carbohydrate transport and metabolism,\u0026quot; and \u0026quot;amino acid transport and metabolism.\u0026quot;\u003c/p\u003e\n\u003cp\u003eIn between-group comparisons, immune system processes were significantly higher in group A compared to groups B and C, likely due to activation in response to infection, allergies, inflammation, or stress. Additionally, \u0026quot;other organism parts\u0026quot; were significantly lower in groups A and F than in the other four groups, possibly due to tissue or organ damage affecting specific functions, such as organ failure, dysfunction, nutritional deficiencies, or aging. These differences in functional profiles between groups A and B-C suggest that the pathogenic \u003cem\u003eE. coli\u003c/em\u003e strains from Shunqing District (group A) had a more significant impact on tissue and organ functions in dogs than those from Gaoping District (group B) or Jialing District (group C) (see Supplemental Data Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4.2 Comparative results of whole gene sequencing virulence factor annotation databases\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Supplemental Data Table 5, whole genome sequencing of pathogenic \u003cem\u003eE. coli\u003c/em\u003e strains and subsequent BLAST database comparison revealed the presence of multiple virulence factors in all six strains. Among these, the most common virulence factors included fimbriae and lipopolysaccharide (LPS). Strains numbered 1-6 and 12-9 exhibited virulence factors related to Brk, while strains numbered 12-4 and 12-1 displayed Ptx. Additionally, Pseudomonas aeruginosa PAO1 was identified as a major shared virulence factor across groups A-C, including lipopolysaccharides, secreted toxins, elastases, histolytic enzymes, and oxidative stress response factors. Burkholderia pseudomallei K96243 and Aeromonas hydrophila ATCC 7966 were also identified as shared sources of multiple virulence genes, with Aeromonas hydrophila having the highest number of virulence genes among all groups. When comparing different groups, the number of virulence genes, including those from Pseudomonas aeruginosa PAO1, was significantly higher in group A compared to groups B and C, suggesting that the \u003cem\u003eE. coli\u003c/em\u003e strain from Nanchong (group A) exhibited virulence resulting from mixed infections with various virulence genes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4.3 Comparison of whole gene sequencing proximate strain databases\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Supplemental Data Figure 3, BLAST comparison of the whole gene sequences of 6 \u003cem\u003eE. coli\u003c/em\u003e strains revealed a close resemblance to \u003cem\u003eE. col\u003c/em\u003ei K-12, \u003cem\u003eE. coli\u003c/em\u003e SE11, \u003cem\u003eE. coli\u003c/em\u003e SE15, and \u003cem\u003eE. coli\u003c/em\u003e 11128. Groups A-C \u003cem\u003eE. coli\u003c/em\u003e strains carried a total of 1614 resistance genes (the major drug resistance genes As shown in Supplemental Data Table 6), primarily associated with antibiotic efflux, alteration of antibiotic targets, and antibiotic target protection. The detection of increased antibiotic efflux expression suggests that antibiotic therapy may be less effective, necessitating alternative therapeutic strategies to combat resistance. The study and monitoring of antibiotic resistance mechanisms in these bacteria are crucial for the development of new antibiotics and for addressing antibiotic-resistant infections.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4.4 Drug sensitivity test results\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe drug sensitivity test was conducted on canine pathogenic \u003cem\u003eE. coli\u003c/em\u003e strains, and the results fell within the acceptable range when compared to established standards for measuring the inhibition zones of various antibiotics. As shown in the figure below, the pathogenic \u003cem\u003eE. coli\u003c/em\u003e strains exhibited a decreasing concentration around the drug sensitivity discs on the agar plate, indicating sensitivity to the drugs. The lack of uniform diffusion in all directions further suggested some degree of resistance to certain antibiotics. The size of the diffusion zone was directly related to both the concentration of the antibiotic discs and the bacterial density.\u003c/p\u003e\n\u003cp\u003eFive strains of \u003cem\u003eE. coli\u003c/em\u003e were randomly tested for resistance to 17 different antibiotics. The results indicated that canine pathogenic \u003cem\u003eE. coli\u003c/em\u003e exhibited varying degrees of resistance to different drugs, as summarized in (supplemental data Table 7). Notably, the strains showed 100% sensitivity to ampicillin, cefazolin, cefoxitin, ceftriaxone, gentamicin, ciprofloxacin, levofloxacin, norfloxacin, doxycycline, chloramphenicol, and others, with moderate resistance observed for some strains to amoxicillin, streptomycin, and butyl carbamate. The varying patterns of resistance suggest that the local frequency and dosage of these antibiotics might influence the observed resistance, confirming the clinical effectiveness of certain drugs (Supplemental Data Figure 4).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we found that the infection rate of canine pathogenic \u003cem\u003eE. coli\u003c/em\u003e disease in the Nanchong area was 3.87% in 2020 and increased to 4.17% in 2021, with an average annual infection rate of 4%. This is a significant increase compared to the 1.6% pathogenic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003einfection rate in the UK, which may be related to the environment or rearing environment [37]. The higher infection of pathogenic \u003cem\u003eE. coli\u003c/em\u003e in the Nanchong area compared to the United Kingdom could be attributed to differences in breeding environments and insufficient attention to the diet of pet dogs by their owners. Additionally, the abundance of animals in Nanchong and the multiple transmission pathways of pathogenic \u003cem\u003eE. coli\u003c/em\u003e contribute to the frequent occurrence of the disease. This elevated prevalence of antimicrobial-resistant \u003cem\u003eE. coli\u003c/em\u003e in domestic dogs may be further contextualized by environmental and behavioral risk factors identified in Canadian settings. A cross-sectional study analyzing dogs frequenting parks in South-Western Ontario found that 23.5% of dogs harbored antimicrobial-resistant \u003cem\u003eE. coli\u003c/em\u003e, with resistance most prevalent against ampicillin (68%), tetracycline (42%), and trimethoprim-sulfamethoxazole (19%) Notably, dogs visiting parks more than twice weekly had 1.8-fold higher odds of carrying resistant strains compared to infrequent visitors, suggesting environmental transmission through fecal-oral routes or direct contact in shared spaces[38]. Although the relative infection rate was stable, there is still a risk of transmission. Significant sex differences were observed in pathogenic E. coli infections, with males exhibiting higher infection rates than females.\u0026nbsp;Teddy, Corgi, and Pomeranian are the commonly infected breeds, and the infection rate of small dogs is higher. Moreover, dogs in the Nanchong area are at risk of infection with pathogenic \u003cem\u003eE. coli\u003c/em\u003e throughout the year, especially in spring and summer; warmer temperatures and increased humidity may be the main causes of the high infection. In this study, the immunization status has little effect on the incidence of \u003cem\u003eE. coli\u003c/em\u003e disease, but immunization remains an important means of preventing infection[39]; the immunized dogs are better able to fight the pathogen and have lower morbidity. In addition, significant differences were found in infection with pathogenic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003ein dogs of different ages. suckling pup and puppies have higher infection rates than adult and older dogs, which may be related to the weaker immunity of puppies. Other studies also found that the younger the animal, the more susceptible to pathogenic \u003cem\u003eE. coli\u003c/em\u003e infection\u0026nbsp;[40]. After follow-up comparative investigation, we found that the living conditions of dogs in Jialing and Gaoping districts are better, and the incidence of pathogenic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eis lower, but the living conditions in Nanbu and Yingshan counties are worse, and the incidence of the disease is relatively higher.Subsequently, we conducted follow-up visits and investigations on dogs living in similar environments and found that living conditions, food, water sources, and the frequency of contact with other animals are the main factors contributing to the infection of pathogenic \u003cem\u003eE. coli\u003c/em\u003e in dogs. When owners restrict their dogs\u0026apos; activity areas to places with less animal waste and lighter soil contamination, the infection rate shows a slight decrease. This indicates that fecal matter and soil pollution are also potential influencing factors. The incidence of pathogenic \u003cem\u003eE. coli\u003c/em\u003e infection in dogs in the Nanchong area is relatively low\u0026nbsp;but still requires attention. Dogs may be susceptible to infection in different seasons and ages, especially in spring and puppies. Symptomatic treatment and the use of antibiotics are key in the treatment process\u0026nbsp;[41]. To effectively prevent and control the occurrence of canine pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e disease, owners should strengthen the management of environmental hygiene\u0026nbsp;[42], boost puppy and suckling pup immunity, monitor pet health, and promptly isolate and treat \u003cem\u003eE. coli\u003c/em\u003e-infected dogs to prevent bacterial spread. When medication was used, it is important to avoid frequent or excessive reliance on antibiotics. The pathogenic rate of \u003cem\u003eE. coli\u003c/em\u003e can be reduced by improving living conditions, reducing contact with unfamiliar animals, and avoiding the consumption of contaminated food and water. For populations with high animal density, it is recommended to vaccinate dogs against pathogenic \u003cem\u003eE. coli\u003c/em\u003e.Veterinarians and public health departments should pay closer attention to the use of antibiotics. Compared to the types of antibiotics previously used, there should be an increased focus on introducing or developing new antibiotics or designing new treatment protocols specifically for pathogenic \u003cem\u003eE. coli\u003c/em\u003e. Additionally, it is important to keep statistics on the frequency of use of various types of antibiotics and the diseases they are used to treat, and to develop strategies to counteract strains similar to tetracycline-resistant bacteria\u0026nbsp;[43].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMany owners in the rearing process, ignore bacterial resistance and antibiotic use, leading to antibiotic abuse, which is the main cause of bacterial resistance [44]. China\u0026apos;s intensive livestock farming has enhanced pathogenic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eadaptability, promoting diversification of virulence factors (e.g., adhesins, toxins) across regions and over time. Analyzing these factors is key to developing targeted interventions against outbreaks.In this study, 27 strains of pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e were obtained through bacterial isolation and culture and 16rs DNA PCR identification from canine faces in the Nanchong area. Six strains were selected for whole gene sequencing. The sequencing results were compared by using blast to analyze the annotation of virulence factors and drug resistance function of pathogenic \u003cem\u003eE. coli\u003c/em\u003e in the Nanchong area, in which the most important virulence factors were\u003cem\u003e\u0026nbsp;Fimbriae\u003c/em\u003e, \u003cem\u003eLPS\u003c/em\u003e, \u003cem\u003eBrk\u003c/em\u003e, \u003cem\u003ePtx, Pertactin\u003c/em\u003e [45].\u0026nbsp;also known as \u003cem\u003eE. coli\u003c/em\u003e bacterial pili. \u003cem\u003eLPS\u0026nbsp;\u003c/em\u003eact as virulence factors, while high-dose\u003cem\u003e\u0026nbsp;LPS\u003c/em\u003e disrupt endothelial barrier function\u0026nbsp;[46]. In a mouse model, \u003cem\u003eBrkA\u003c/em\u003e mutants showed attenuated respiratory tract infectivity compared to wild-type strains. These mutants also exhibited increased complement sensitivity in vitro, confirming \u003cem\u003eBrkA\u0026apos;s\u003c/em\u003e role in resisting complement-mediated killing. Moreover, multiple \u003cem\u003eDeltabrkA\u003c/em\u003e mutants derived from Tohama I in mice exhibited severe defects within the first week after inoculation. This defect was present even in complement-deficient mice, revealing a complement-independent phenotype of \u003cem\u003eBrkA\u003c/em\u003e in respiratory tract infections[47]\u0026nbsp;\u003cem\u003ePTX\u003c/em\u003e, utilizing its unique ADP-ribosylation capability, disrupts and perturbs the intricate signaling processes within cells. This interference impedes the normal functioning of cells, leading to dysfunction and potentially triggering cell death. In essence, \u003cem\u003ePTX\u003c/em\u003e impairs host cell signaling\u0026nbsp;[48], while pertactin mediates B. pertussis epithelial adhesion and immune escape via expression plasticity, sustaining infection\u0026nbsp;[49].\u003c/p\u003e\n\u003cp\u003eThe isolates of pathogenic \u003cem\u003eE. coli\u003c/em\u003e had certain drug resistance, mainly expressed as antibiotic efficacy. All the strains have some sensitivity to antibiotics through drug sensitivity tests; tetracycline sensitivity is the lowest, at 60%, followed by amoxicillin, streptomycin, and butamocarbamol, with a sensitivity of 80%. Most of the\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e colonization factors are expressed as adhesins. A study conducted on canine\u003cem\u003e\u0026nbsp;E. coli\u0026nbsp;\u003c/em\u003eisolates from clinical samples in the northeastern U.S. revealed resistance rates ranging from 0.4% (amikacin) to 34.3% (ampicillin). Survey data indicated significant increasing trends in resistance to several drugs, including cephalosporins, enrofloxacin, and tetracycline. Similarly, Cummings\u0026apos; study highlighted widespread AMR and MDR in pathogenic \u003cem\u003eE. coli\u003c/em\u003e from both dogs and cats, largely attributed to extensive antimicrobial use in veterinary practice. High resistance rates were observed against \u0026beta;-lactams, fluoroquinolones, and sulfonamides, with notable regional variations in the resistance profiles[50]. On \u003cem\u003eE. coli\u003c/em\u003e isolates from clinical canine samples in New York, USA, revealed a significant increase in MDR among the bacterial strains due to the extensive use of antibiotics and other medications. Specifically, MDR was prevalent, with high resistance rates to ampicillin and cephalosporins, showing increasing trends over time. Resistance to gentamicin and enrofloxacin also rose progressively, while sulfonamides exhibited persistently high resistance [51]. Therefore, it can be seen that the antibiotic resistance of pathogenic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003einfections in dogs varies from region to region. Antibiotic efflux pumps are key in bacterial resistance, expelling antibiotics from cells to lower their intracellular concentration and allow bacterial survival. Antibiotic efflux pumps are major contributors to enhanced bacterial resistance by expelling antibiotics from cells to reduce drug concentration, allowing bacteria to combat antibiotics [52]. Mutations in bacterial genomes can alter antibiotic target proteins, reducing drug binding affinity or disrupting their function, thereby conferring resistance. This adaptive evolution is a key driver of resistance development. Furthermore, mobile genetic elements (e.g., plasmids, transposons) enable cross-species dissemination of resistance genes through conjugation or transposition, accelerating resistance spread. These vectors act as \u0026quot;resistance propagators,\u0026quot; rapidly dispersing resistance traits across bacterial populations[53]. Notably, Bacterial biofilms act as protective barriers, impeding antibiotic penetration. Within biofilms, bacteria exhibit reduced metabolic activity and antibiotic susceptibility. The biofilm matrix (e.g., polysaccharides, proteins) further neutralizes antimicrobial agents. Of public health concern, pathogenic \u003cem\u003eE. coli\u003c/em\u003e resistance genes can transfer via mobile genetic elements to Salmonella or commensal strains, accelerating resistance dissemination under antimicrobial pressure [54]. Consequently, biofilm formation represents another pivotal strategy bacteria employ to withstand antibiotic assault. In conclusion, antibiotic efflux pumps, coupled with bacterial genomic mutations, interbacterial genetic exchange, and biofilm formation, constitute a complex network underpinning bacterial resistance. Understanding these mechanisms is paramount for developing novel antibiotics, optimizing existing treatment protocols, and formulating effective resistance prevention and control strategies.\u003c/p\u003e\n\u003cp\u003eThe rampant abuse of antibiotics in China has become a pressing issue, significantly exacerbated the spread of bacterial resistance, and compromise the efficacy of antibiotics as a \u0026quot;lifesaving shield,\u0026quot; rendering numerous once-manageable infectious diseases more complex and difficult to treat. To effectively combat health threats like canine colibacillosis, it\u0026apos;s crucial to regulate antibiotic use in pet hospitals for precise medication. Rational antibiotic use and reducing unnecessary prescriptions are key to curbing antibiotic resistance. Concurrently, the urgent need for novel antibiotic development is paramount in the face of growing resistance challenges. These new drugs must possess novel targets and mechanisms of action, capable of penetrating existing resistance barriers and paving the way for innovative treatment strategies against resistant pathogens. Furthermore, we must actively explore and promote alternative therapies such as immunotherapy and bacteriophage therapy, which not only diversify treatment options for resistant infections but also alleviate the pressure on antibiotic usage, further hindering the progression of resistance. We must adopt a multifaceted approach to tackle the challenge of antibiotic misuse by strengthening regulation, promoting rational use, developing new drugs, advocating alternative therapies, and enhancing monitoring and prevention, thus safeguarding the health of humans and animals and ensuring the sustainable use of antibiotics in medical treatment.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, canine pathogenic \u003cem\u003eE. coli\u003c/em\u003e disease has a large negative impact on the overall health status of dogs and social security and health. This study investigates the epidemiological pathogenicity of domestic dogs in the Nanchong area and isolation of pathogenic \u003cem\u003eE. coli\u003c/em\u003e to analyze the whole gene, focusing on the virulence factor and its resistance to complete the in-depth analysis, the preliminary confirmation of the current status of the rate of infection pathogenic\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e and its strains of pathogenicity. The epidemiological status of pathogenic \u003cem\u003eE. coli\u003c/em\u003e was shown in Figure 4.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Chuanbei Animal Hospital in Nanchong City for their support of this research. In addition, we would like to acknowledge all the animal hospitals and pet owners who allowed the use of samples in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBangyuan Wu: Conceptualization, Formal analysis, Data curation, Validation. Kai Li: Formal analysis, Writing-original draft, Data curation. Aifei Du: Formal analysis, Data curation. Shunjie Tang: Conceptualization, Formal analysis. Shaohua Feng: Conceptualization, Supervision. Shibin Yuan: Supervision, Conceptualization, Methodology, Writing-Review \u0026amp; Editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was fully supported by the Nanchong Key Laboratory of Wildlife Nutrition Ecology and Disease Control, Sichuan, China (NCKL202201), the National Natural Science Foundation of China (32370557), Key Project of the Joint Fund for Science and Education of Sichuan Province(2024NSFSC1967) and the Fundamental Research Funds of China West Normal University (Project No. 20A003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval the study was conducted in accordance with the guidelines established by the International Animal Ethics Committee, and the treatment of the animals adhered to the guidelines set forth by the Animal Care Committee of China West Normal University (2024LLSC0052). The environmental conditions and facilities for the animals met the national standards\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePakbin B, Br\u0026uuml;ck W M, Rossen J W. Virulence factors of enteric pathogenic Escherichia coli: A review. International journal of molecular sciences.2021;22(18): 9922.\u003c/li\u003e\n\u003cli\u003eSheikh A, Fleckenstein J M. Interactions of pathogenic Escherichia coli with CEACAMs. Frontiers in immunology.2023;14:1120331.\u003c/li\u003e\n\u003cli\u003eClements A, Young J C, Constantinou N, Frankel G. Infection strategies of enteric pathogenic Escherichia coli. 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Frontiers in microbiology.2013;4(258.\u003cu\u003e\u003c/u\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dog, Drug resistance, Epidemiology Toxicity factors, Nanchong, Pathogenic Escherichia coli, Whole genome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-7708388/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7708388/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Pathogenic Escherichia coli is a zoonotic bacterial pathogen that causes significant losses to the farming industry and threatens public health safety. Dogs, as companion animals, are closely intertwined with human life and health. Therefore, heightened attention to pathogenic E. coli in canines is particularly crucial. This study investigated the infection and incidence of canine pathogenic E. coli disease in Nanchong of Sichuan province in China. 3229 diseased dogs from 2020 to 2021, according to different seasons, gender, age, breed, and immunization status, were analyzed. Meanwhile, pathogenic E. coli was isolated and identified from the fecal samples of the sick dogs, and the virulence factors and drug resistance of the strains were also analyzed. The study identified 129 cases of pathogenic E. coli infections in dogs from 2020 to 2021, accounting for 4% of the total (129/3229). compared to factors such as season, sex, and immune status, there is a significant difference in the infection rates between different age groups of dogs (P=0.029). Canine pathogenic E. coli can be infected throughout the year, The study found that the highest rate of pathogenic E. coli infections in dogs occurred during spring, with summer having the second-highest rate, followed by winter. The infection rate of males was higher than that of females, the infection rate of juvenile dogs was higher than that of adult dogs, and the infection rate of small dogs was higher than that of medium-sized and large dogs. In addition, all 27 pathogenic E. coli strains isolated from diverse geographical regions demonstrated antibiotic susceptibility, with the lowest sensitivity to tetracycline at 60%, followed by amoxicillin, streptomycin, and butyl carbamate, with a sensitivity of 80%. The results of whole gene sequencing showed that the main virulence factors were Fimbriae, LPS, Brk, and the canine pathogenic E. coli in the Nanchong area had developed resistance to some antibiotics. Although the infection rate of pathogenic E. coli in canines was relatively low in Nanchong, it remains essential to advance research on its pathogenic characteristics, antimicrobial resistance patterns, and control measures. Concurrently, clinical antimicrobial use must be standardized, immunity in juvenile dogs enhanced, and environmental hygiene management within breeding facilities strengthened.","manuscriptTitle":"Epidemiologic investigation of pathogenic Escherichia coli in domestic dogs in Nanchong area","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 05:40:29","doi":"10.21203/rs.3.rs-7708388/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"524a7224-2602-4218-91d6-15efe3d7aaaf","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-12T11:08:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-10 05:40:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7708388","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7708388","identity":"rs-7708388","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}