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Abstract [Objectives]The study aimed to identify the pathogenic E. coli strain that caused diarrhea in foxes and to analyze its drug sensitivity.
[Methods]A pathogenic E. coli strain was isolated from dead foxes with diarrhea. By conventional bacterial isolation and culture， morphological observation， pathogenicity test and KB disc method， the isolated strain was identified as pathogenic E. coli.
[Results]The isolated pathogen was highly sensitive to ceftriaxone， cefotaxime， ciprofloxacin and lincomycin， moderately sensitive to enrofloxacin， neomycin， gentamycin， spectinomycin， florfenicol， amikacin and polymyxin， and resistant to ampicillin， amoxicillin and doxycycline.
[Conclusions]This study provided reference for the prevention and control of diarrheal diseases in foxes in Qinhuangdao region.
Key words Fox； Diarrhea； Pathogenic E. coli； Drug sensitivity test
Based on hereditary and clinical symptoms， Escherichia coli can be divided into intraintestinal pathogenic E. coli， extraintestinal pathogenic E. coli and symbiotic E. coli， among which intraintestinal pathogenic E. coli and extraintestinal pathogenic E. coli are important zoonotic pathogens that may cause various diseases in chickens， ducks， pigs， sheep， minks， foxes and many other animals， resulting in serious local or systemic infections[1-2]. In foxes， pathogenic E. coli mainly infects pre and postweaning cubs， leading to epidemic outbreaks with certain seasonality. The main clinical symptoms are sepsis and diarrhea， with relatively high incidence and mortality. In addition， pathogenic E. coli can also invade the central nervous system and respiratory system. Adult and old foxes are rarely affected， whereas pathogenic E. coli infections in adult female foxes may cause miscarriage and stillbirth， resulting in serious economic losses[3-5].
In May 2018， 10 out of 5 000 foxes had severe diarrhea in a fox breeding farm in Qinhuangdao， and 2 foxes died. The foxes died of diarrhea were necropsied. A pathogenic E. coli strain was isolated from aseptically collected liver tissues for identification， pathogenicity test and drug sensitivity assay. The results provided reference for the prevention and control of diarrheal diseases in foxes in Qinhuangdao region.
Materials and Methods
Pathological materials
In May 2018， among 5 000 foxes in a fox breeding farm in Qinhuangdao， 10 foxes had severe diarrhea and 2 foxes died. The foxes died of diarrhea were necropsied. The liver tissues were aseptically collected for pathogen isolation and identification. Reagents and instruments
Eosinmethylene blue （EMB） medium， MaConkey agar medium and nutrient broth were purchased from Qingdao Hope BioTechnology Co.， Ltd.； drug sensitive slips and bacteria identification test paper were purchased from Beijing Tiantan Biological Products Co.， Ltd.； ID32E intestinal bacteria identification test paper was produced by Biomérieux Inc. （France）； waterjacket thermostatic incubator was produced by Shanghai Yiheng Scientific Instruments Co.， Ltd.； conventional instruments and reagents were provided by Key Laboratory of Preventive Veterinary Medicine of Hebei Province.
Experimental animals
Ten healthy Kunming mice， weighing about 22 g， were purchased from Beijing Vital River Laboratory Animal Technology Co.， Ltd.
Isolation and identification of the pathogenic strain
Liver tissues were aseptically collected from foxes died of diarrhea， inoculated to MaConkey agar medium and incubated at 37 ℃ for 12-18 h. The dominant colonies were inoculated to eosinmethylene blue medium and incubated at 37 ℃ for 12-18 h. A single colony was inoculated to nutrient broth for purification culture. The morphological characteristics were observed by Gram staining and microscopy.
Biochemical test
Biochemical characteristics of the isolated strain were analyzed according to the introductions. The isolated strain was identified by using ATB automatic biochemical identification system.
Pathogenicity test
Ten Kunming mice were divided into two groups， five mice per group. Each mouse in the experimental group was administered orally 0.25 ml （109 cfu/ml） of the isolated strain. Mice in the control group were given an equal amount of sterile saline. After 12 hours， mice in different groups were observed for 7 days. The incidence and mortality of mice in each group were recorded. The pathogenic strain in dead mice was isolated and identified.
Drug sensitivity test
Drug sensitivity of the isolated strain was analyzed and evaluated according to the standard KB disc method recommended by the National Committee for Clinical Laboratory Standards of America （NCCLS）. Under aseptic conditions， the concentration of bacterial liquid of 20 extraintestinal pathogenic E. coli strains from furbearing animals was adjusted to 0.5 MCF； 100 μl of bacterial liquid was coated evenly on the common culture medium. After the bacterial liquid was completely absorbed， drug sensitive slips were stuck to the medium. Each drug sensitivity test was repeated three times， and the results were averaged. The medium was incubated at 37 ℃ for 8-12 h. The diameter of inhibition zone was measured with a ruler to determine the drug sensitivity. Results and Analysis
Isolation， purification and morphological observation of the pathogenic strain
The isolated strain exhibited moist， oblate， pink colonies with smooth edge on MaConkey agar medium， and deep purple circular colonies with metallic luster on eosinmethylene blue medium. Gram staining and microscopy indicated that the isolated strain HBE2 was a Gramnegative short rodshaped bacterium with obtuse ends.
Biochemical test
The results of biochemical test showed that the isolated strain could produce acid without producing gas by fermenting mannitol， glucose， trehalose， maltose， arabinose and rhamnose， but it could not decompose urea. The indole test exhibited positive results， whereas the VP test exhibited negative results. By using ATB automatic biochemical identification system， the isolated strain was identified as E. coli， with a qualification rate of 99.9%.
Pathogenicity test
Mice in the experimental group died at around 24-48 h， with a mortality of 100%. A pathogenic strain was isolated from the dead mice. Five mice in the control group were healthy and survived， indicating that the isolated E. coli strain had strong pathogenicity to mice.
Drug sensitivity test
As could be seen in Table 1， the pathogenic E. coli strain isolated from foxes was highly sensitive to ceftriaxone， cefotaxime， ciprofloxacin and lincomycin， moderately sensitive to enrofloxacin， neomycin， gentamycin， spectinomycin， florfenicol， amikacin and polymyxin， and resistant to ampicillin， amoxicillin and doxycycline.
Conclusion and Discussion
Pathogenic E. coli is an important zoonotic pathogen that can cause diseases in chickens， ducks， pigs， sheep， minks， foxes and many other animals， and lead to serious local or systemic infections. Moreover， it can also cause various organ infections in minks and foxes， such as pneumonia， sepsis， meningitis and urinary tract infections， with relatively high incidence and mortality[6-7]. In this study， a pathogenic strain was isolated from foxes died of diarrhea and inoculated to Kunming mice artificially. The results showed that the isolated strain was highly pathogenic to mice， which confirms that pathogenic E. coli is the main pathogen that causes diarrhea in foxes. Pathogenic E. coli may pose a potential threat to foxes and should be paid much attention to. The results of drug sensitivity test indicated that the pathogenic strain isolated from foxes died of diarrhea was highly sensitive to ceftriaxone， cefotaxime， ciprofloxacin and lincomycin， moderately sensitive to enrofloxacin， neomycin， gentamycin， spectinomycin， florfenicol， amikacin and polymyxin， and resistant to ampicillin， amoxicillin and doxycycline. In order to prevent drug resistance， antibiotics should be used reasonably after drug sensitivity test to improve the therapeutic effect against the pathogenic strain. References
[1]ZHANG ZX. Application of Chinese herbal medicine ultrafine powder in prevention and treatment of pathogenic E. coli from chicken[D]. Qinhuangdao： Hebei Normal University of Science and Technology， 2017.（in Chinese）
[2]XIA QQ， WEN SS， MA SQ， et al. Detection and analysis of biofilm formation ability， antimicrobialresistance and pathogenic island gene of pathogenic Escherichia coli isolated from minks[J]. Chinese Journal of Preventive Veterinary Medicine， 2017， 39（5）：361-365.（in Chinese）
[3]ZHANG ZX， XIAO LR， LI QL， et al. Antibacterial effects of 26 kinds of Chinese herbal medicines on pathogenic E. coli from foxes in vitro[J]. Progress in Veterinary Medicine， 2018， 39（2）：127-130.（in Chinese）
[4]ZHANG ZQ， WU TL， LI YH， et al. Prokaryotic expression and polyclonal antibody preparation of HtrA protein from Escherichia coli causing pneumonia in foxes[J]. Chinese Journal of Veterinary Science， 2017， 37（7）：1255-1260.（in Chinese）
[5]ZHANG ZX， LI YY， ZHANG YY， et al. Isolation， identification and drug sensitivity analysis of E. coli from raccoon dogs in Qinhuangdao[J]. Heilongjiang Animal Science and Veterinary Medicine， 2016， （12）：175-177.（in Chinese）
[6]RUI P， MA ZJ， YANG CR， et al. Isolation and identification of pathogenic Escherichia coli from foxes， raccoon dogs and minks[J]. Chinese Veterinary Science， 2015， 45（5）：517-521.（in Chinese）
[7]YU J， QU HN， ZHANG C， et al. Resistance of E. coli from furbearing animals to fluoroquinolones and compound Chinese medicine[J]. China Animal Husbandry & Veterinary Medicine， 2015， 42（6）：1597-1601.（in Chinese）
Editor： Chunmei WU Proofreader： Xinxiu ZHU
[Methods]A pathogenic E. coli strain was isolated from dead foxes with diarrhea. By conventional bacterial isolation and culture， morphological observation， pathogenicity test and KB disc method， the isolated strain was identified as pathogenic E. coli.
[Results]The isolated pathogen was highly sensitive to ceftriaxone， cefotaxime， ciprofloxacin and lincomycin， moderately sensitive to enrofloxacin， neomycin， gentamycin， spectinomycin， florfenicol， amikacin and polymyxin， and resistant to ampicillin， amoxicillin and doxycycline.
[Conclusions]This study provided reference for the prevention and control of diarrheal diseases in foxes in Qinhuangdao region.
Key words Fox； Diarrhea； Pathogenic E. coli； Drug sensitivity test
Based on hereditary and clinical symptoms， Escherichia coli can be divided into intraintestinal pathogenic E. coli， extraintestinal pathogenic E. coli and symbiotic E. coli， among which intraintestinal pathogenic E. coli and extraintestinal pathogenic E. coli are important zoonotic pathogens that may cause various diseases in chickens， ducks， pigs， sheep， minks， foxes and many other animals， resulting in serious local or systemic infections[1-2]. In foxes， pathogenic E. coli mainly infects pre and postweaning cubs， leading to epidemic outbreaks with certain seasonality. The main clinical symptoms are sepsis and diarrhea， with relatively high incidence and mortality. In addition， pathogenic E. coli can also invade the central nervous system and respiratory system. Adult and old foxes are rarely affected， whereas pathogenic E. coli infections in adult female foxes may cause miscarriage and stillbirth， resulting in serious economic losses[3-5].
In May 2018， 10 out of 5 000 foxes had severe diarrhea in a fox breeding farm in Qinhuangdao， and 2 foxes died. The foxes died of diarrhea were necropsied. A pathogenic E. coli strain was isolated from aseptically collected liver tissues for identification， pathogenicity test and drug sensitivity assay. The results provided reference for the prevention and control of diarrheal diseases in foxes in Qinhuangdao region.
Materials and Methods
Pathological materials
In May 2018， among 5 000 foxes in a fox breeding farm in Qinhuangdao， 10 foxes had severe diarrhea and 2 foxes died. The foxes died of diarrhea were necropsied. The liver tissues were aseptically collected for pathogen isolation and identification. Reagents and instruments
Eosinmethylene blue （EMB） medium， MaConkey agar medium and nutrient broth were purchased from Qingdao Hope BioTechnology Co.， Ltd.； drug sensitive slips and bacteria identification test paper were purchased from Beijing Tiantan Biological Products Co.， Ltd.； ID32E intestinal bacteria identification test paper was produced by Biomérieux Inc. （France）； waterjacket thermostatic incubator was produced by Shanghai Yiheng Scientific Instruments Co.， Ltd.； conventional instruments and reagents were provided by Key Laboratory of Preventive Veterinary Medicine of Hebei Province.
Experimental animals
Ten healthy Kunming mice， weighing about 22 g， were purchased from Beijing Vital River Laboratory Animal Technology Co.， Ltd.
Isolation and identification of the pathogenic strain
Liver tissues were aseptically collected from foxes died of diarrhea， inoculated to MaConkey agar medium and incubated at 37 ℃ for 12-18 h. The dominant colonies were inoculated to eosinmethylene blue medium and incubated at 37 ℃ for 12-18 h. A single colony was inoculated to nutrient broth for purification culture. The morphological characteristics were observed by Gram staining and microscopy.
Biochemical test
Biochemical characteristics of the isolated strain were analyzed according to the introductions. The isolated strain was identified by using ATB automatic biochemical identification system.
Pathogenicity test
Ten Kunming mice were divided into two groups， five mice per group. Each mouse in the experimental group was administered orally 0.25 ml （109 cfu/ml） of the isolated strain. Mice in the control group were given an equal amount of sterile saline. After 12 hours， mice in different groups were observed for 7 days. The incidence and mortality of mice in each group were recorded. The pathogenic strain in dead mice was isolated and identified.
Drug sensitivity test
Drug sensitivity of the isolated strain was analyzed and evaluated according to the standard KB disc method recommended by the National Committee for Clinical Laboratory Standards of America （NCCLS）. Under aseptic conditions， the concentration of bacterial liquid of 20 extraintestinal pathogenic E. coli strains from furbearing animals was adjusted to 0.5 MCF； 100 μl of bacterial liquid was coated evenly on the common culture medium. After the bacterial liquid was completely absorbed， drug sensitive slips were stuck to the medium. Each drug sensitivity test was repeated three times， and the results were averaged. The medium was incubated at 37 ℃ for 8-12 h. The diameter of inhibition zone was measured with a ruler to determine the drug sensitivity. Results and Analysis
Isolation， purification and morphological observation of the pathogenic strain
The isolated strain exhibited moist， oblate， pink colonies with smooth edge on MaConkey agar medium， and deep purple circular colonies with metallic luster on eosinmethylene blue medium. Gram staining and microscopy indicated that the isolated strain HBE2 was a Gramnegative short rodshaped bacterium with obtuse ends.
Biochemical test
The results of biochemical test showed that the isolated strain could produce acid without producing gas by fermenting mannitol， glucose， trehalose， maltose， arabinose and rhamnose， but it could not decompose urea. The indole test exhibited positive results， whereas the VP test exhibited negative results. By using ATB automatic biochemical identification system， the isolated strain was identified as E. coli， with a qualification rate of 99.9%.
Pathogenicity test
Mice in the experimental group died at around 24-48 h， with a mortality of 100%. A pathogenic strain was isolated from the dead mice. Five mice in the control group were healthy and survived， indicating that the isolated E. coli strain had strong pathogenicity to mice.
Drug sensitivity test
As could be seen in Table 1， the pathogenic E. coli strain isolated from foxes was highly sensitive to ceftriaxone， cefotaxime， ciprofloxacin and lincomycin， moderately sensitive to enrofloxacin， neomycin， gentamycin， spectinomycin， florfenicol， amikacin and polymyxin， and resistant to ampicillin， amoxicillin and doxycycline.
Conclusion and Discussion
Pathogenic E. coli is an important zoonotic pathogen that can cause diseases in chickens， ducks， pigs， sheep， minks， foxes and many other animals， and lead to serious local or systemic infections. Moreover， it can also cause various organ infections in minks and foxes， such as pneumonia， sepsis， meningitis and urinary tract infections， with relatively high incidence and mortality[6-7]. In this study， a pathogenic strain was isolated from foxes died of diarrhea and inoculated to Kunming mice artificially. The results showed that the isolated strain was highly pathogenic to mice， which confirms that pathogenic E. coli is the main pathogen that causes diarrhea in foxes. Pathogenic E. coli may pose a potential threat to foxes and should be paid much attention to. The results of drug sensitivity test indicated that the pathogenic strain isolated from foxes died of diarrhea was highly sensitive to ceftriaxone， cefotaxime， ciprofloxacin and lincomycin， moderately sensitive to enrofloxacin， neomycin， gentamycin， spectinomycin， florfenicol， amikacin and polymyxin， and resistant to ampicillin， amoxicillin and doxycycline. In order to prevent drug resistance， antibiotics should be used reasonably after drug sensitivity test to improve the therapeutic effect against the pathogenic strain. References
[1]ZHANG ZX. Application of Chinese herbal medicine ultrafine powder in prevention and treatment of pathogenic E. coli from chicken[D]. Qinhuangdao： Hebei Normal University of Science and Technology， 2017.（in Chinese）
[2]XIA QQ， WEN SS， MA SQ， et al. Detection and analysis of biofilm formation ability， antimicrobialresistance and pathogenic island gene of pathogenic Escherichia coli isolated from minks[J]. Chinese Journal of Preventive Veterinary Medicine， 2017， 39（5）：361-365.（in Chinese）
[3]ZHANG ZX， XIAO LR， LI QL， et al. Antibacterial effects of 26 kinds of Chinese herbal medicines on pathogenic E. coli from foxes in vitro[J]. Progress in Veterinary Medicine， 2018， 39（2）：127-130.（in Chinese）
[4]ZHANG ZQ， WU TL， LI YH， et al. Prokaryotic expression and polyclonal antibody preparation of HtrA protein from Escherichia coli causing pneumonia in foxes[J]. Chinese Journal of Veterinary Science， 2017， 37（7）：1255-1260.（in Chinese）
[5]ZHANG ZX， LI YY， ZHANG YY， et al. Isolation， identification and drug sensitivity analysis of E. coli from raccoon dogs in Qinhuangdao[J]. Heilongjiang Animal Science and Veterinary Medicine， 2016， （12）：175-177.（in Chinese）
[6]RUI P， MA ZJ， YANG CR， et al. Isolation and identification of pathogenic Escherichia coli from foxes， raccoon dogs and minks[J]. Chinese Veterinary Science， 2015， 45（5）：517-521.（in Chinese）
[7]YU J， QU HN， ZHANG C， et al. Resistance of E. coli from furbearing animals to fluoroquinolones and compound Chinese medicine[J]. China Animal Husbandry & Veterinary Medicine， 2015， 42（6）：1597-1601.（in Chinese）
Editor： Chunmei WU Proofreader： Xinxiu ZHU