Study on the Therapeutic Effect of Modified Yujin Powder on E. coli in the Procapra przewalskii

Chunjie Song1, Shangquan Gan2 and Xiaoyun Shen1,3*

1School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China

2State Key Laboratory of Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi, Xinjiang 832000, China

3World Bank Poverty Alleviation Project Office in Guizhou, Southwest China, Guiyang 550004, China

ABSTRACT

This experiment was to study the therapeutic effect of modified Yujin powder on E. coli in the P. przewalskii. Twenty the Procapra przewalskii with the E. coli were selected from the Wildlife Rescue Center of the Qinghai Lake National Nature Reserve and randomly divided into low-dose group, high-dose group, gentamicin sulfate group and control group with 5 Procapra przewalskii in each group. The P. przewalskii in modified Yujin powder group were given Yujing powder once every morning and evening, 3, 4 mL/ (kg·BW) for the low-dose group and high-dose group, respectively. The P. przewalskii were injected with gentamicin sulfate intramuscularly,once every morning and evening. The P. przewalskii in control group were fed normally and no medicine was given. After 7 d, the clinical symptoms of the P. przewalskii were observed, and the blood routine, serum biochemical parameter, serum immune and antioxidant indices were measured. The results showed that the P. przewalskii were sensitive reaction, increased appetite, and no diarrhea in the modified Yujin powder group. The healing rates were 75%, 85%, and 60% in low-dose group, high-dose group, gentamicin sulfate group, respectively. The effects of modified Yujin powder was better than gentamicin sulfate. The contents of blood urea nitrogen (BUN), malondialdehyde (MDA), and the number of white blood cell (WBC) in 3 drug delivery groups were extremely significantly lower (P<0.01) than the control group, and the contents of potassium (K), total protein (TP), albumin (ALB), globulin (GLB), and total antioxidant capacity (T-AOC) in the 3 drug delivery groups were extremely significantly higher (P<0.01) than that of control group, but there were no remarkable differences between the 3 drug delivery groups. The serum catalase (CAT) activity in the high-dose group was extremely significantly higher (P<0.01) than that in the low-dose group, gentamicin sulfate group, and control group. Consequently, modified Yujin powder had a good therapeutic effect on the E. coli in P. przewalskii, and the effect of the high-dose group was better.

Article Information

Received 25 November 2019

Revised 22 February 2020

Accepted 04 March 2020

Available online 09 April 2021

(early access)

Published 25 January 2022

Authors’ Contribution

CJS collected material, compiled the results and writing of manuscript. CJS and SQG performed the experiments. XYS reviewed the manuscript.

Key words

The E. coli, The P. przewalskii, Modified Yujin powder, Gentamicin sulfate, Blood parameter

DOI: https://dx.doi.org/10.17582/journal.pjz/20191125111151

* Corresponding author: [email protected]

0030-9923/2022/0002-0859 $ 9.00/0

Copyright 2022 Zoological Society of Pakistan

INTRODUCTION

The Przewalski’s gazelle (Procapra przewalskii) is endemic to the Qinghai-Tibetan Plateau, China, and has been widely distributed throughout the Inner Mongolia, Ningxia, Gansu, Qinghai, Xinjiang, and Tibet regions (Hu and Jiang, 2011; Ping et al., 2018). In recent years, the distribution range of the P. przewalskii has been gradually shrinking because of human population growth, grassland degradation, and habitat fragmentation (Shen et al., 2018; Chi et al., 2020; Jiang, 2004). Currently, only the Qinghai Lake basin remains preserved as a habitat for the P. przewalskii (Yang et al., 2006). According to the survey results in recent years, only the P. przewalskii populations comprising about 300 animals inhabit the Qinghai Lake area (Zhang et al., 2018). Therefore, the P. przewalskii has become the least populous species among the endemic mammals in China (Shen et al., 2009, 2019; Jiang and Wang, 2000. In 2008, the P. przewalskii was recategorized on the International Union for Conservation of Nature (IUCN) Red List from “critically endangered” to “endangered” (Li et al., 2012).

The colibacillary (Escherichia coli) is an acute bacterial infection caused by infection with pathogenic E. coli (Fernando et al., 2019; Wu, 2014). It is characterized by depression, loss of appetite, abnormal growth and development, diarrhea, and high mortality (Fit et al., 2019; Mainil, 2013). The main characteristics of affected animals are to excrete the feces with foul smell and gray color (Ma, 2017). The disease will not only cause dehydration and debilitation, but also increase the mortality of P. przewalskii. At present, the treatment of E. coli is mainly based on antibiotics, but there are problems such as drug residues and drug resistance (Su et al., 2019). In recent years, the research and development of Chinese herbal medicine antibacterial and antiviral drugs has become a research hot spot at domestic and abroad (Tong et al., 2014). Traditional Chinese medicine can not only directly kill bacteria, but also exert antibacterial effect by enhancing the organism immunity. Traditional Chinese medicine has the dual functions of medicinal and nutrient in clinical practice, which is environmentally friendly, safe and resistant to drug resistance (Ning et al., 2015; Ho et al., 2013). Chinese herbal medicines have small side effects, low prices, and wide sources, which can also reduce the cost of breeding (Tong et al., 2014; Meng et al., 2015).

According to the Chinese veterinary syndrome theory, E. coli is caused by damp-heat hoarding of the intestinal tract, which should be heat-clearingand detoxifying, eliminating dampness and relieving diarrhea (Li, 2017). Yujin powder is the prescription in “Yuan Heng Liao Ma Ji” (Commission of Chinese veterinary pharmacopoeia, 2011). It has the effects of heat-clearingand detoxifying, dispersing phlegm and stopping diarrhea (Liao et al., 1990). This experiment adds 4 Chinese herbal medicines of Atractylodes macrocephala, Glycyrrhiza uralensis, Common vladimiria and Pulsatilla chinensis on the basis of Yujin powder, and subtracts Scutellaria baicalensis, Phellodendron amurense, Gardenia jasminoides and Rheum palmatum. Take Yujin to promote and normalize the flow of qi as a principle drug. Atractylodes macrocephala, Glycyrrhiza uralensis, and Common vladimiria can regulating qi for invigorating spleen, as an adjuvant drug. Coptis chinensis and Pulsatilla chinensis can clear heat, eliminating dampness, and removing toxicity, as an assistant drug. Terminalia chebula relieving diarrhea with astringents. Cynanchum otophyllum retaining yin with astringent and harmonizing method (Li, 2002). The combination of various drugs has the effects of clearing away heat and detoxifying, relieving diarrhea with astringents. By studying the therapy of modified Yujin powder on E. coli in P. przewalskii, it aims to provide a reference for clinical treatment of such bacterium, and also provides new ideas for the protection of the P. przewalskii.

MATERIALS AND METHODS

Preparation of traditional Chinese medicine and gentamicin sulfate

Modified Yujin powder is composed of tulip, S. baicalensis and G. uralensis, each 25 g. A. macrocephala, C. vladimiria, P. chinensis, and C. otophyllum, each 20 g, and T. chebula 15 g. Soak for 30 min with water, boil quickly with a fire, then use a simmer to cook. The drug juice was filtered, and the filtered drug residue and the appropriate amount of water were further boiled for 30 min, and then filtered, and the filtrate was combined and concentrated to contain 1 g of crude drug per 1 mL of the drug solution.

Experimental animals

The experimental animals were selected from the Wildlife Rescue Center of the Qinghai Lake National Nature Reserve. E. coli antibody test was carried out on P. przewalskii, and twenty animals (1.5-year-old) with negative antibody detection and clinical symptoms were selected. Nutrition level of grassland types in the habitat of P. przewalskii as shown in Table I.

 

Table I. Nutrition level in main grassland types of P. przewalskii (%).

Category	CP	EE	CF	NFE
Artemisia desertorum shrubland	11.36	3.86	30.88	33.94
Agropyron cristatum grassland	9.16	2.36	35.63	45.99
Iris lacteal grassland	12.10	2.81	29.00	33.70
Achnatherum splendens grassland	10.96	2.94	26.06	49.18
Potentilla discolor grassland	14.89	3.06	18.17	47.53
Stipa purpursa grassland	13.10	3.00	27.89	42.52
Elymus nutans grassland	9.90	2.73	28.37	48.65
Carex rigescens grassland	11.44	2.80	28.35	45.27
Orinus kokonorica grassland	14.98	4.07	21.05	41.17
Stipa breviflora grassland	13.80	3.61	28.28	38.36

 

CP, crude protein; EE, ether extract; CF, crude fiber; NFE, nitrogen free extract.

 

Experimental design

Twenty P. przewalskii with E. coli were randomly divided into 4 groups. Modified Yujin powder were low-dose group and high-dose group. According to the low (3 mL/kg, BW), high (4 mL/kg, BW) dose of intragastric administration, each morning and evening, dosing for 7 d. The gentamicin sulfate group intramuscular injection of 10 mL gentamicin sulfate 0.1-0.2 mL/kg, twice a day, for 7 d. The control group was fed normally and was not administered. The test period was 14 days, and the drug was administered for 7 d, observe for 7 d.

Judgment of drug effect: After healing, the clinical symptoms of the affected animals completely disappeared, the affected animals were react quickly, with a strong appetite, no diarrhea, and no recurrence within 7 d after stopping the drug. Effective is that the main clinical symptoms of the affected animals, has a good mental state, increased appetite, and reduced diarrhea, but relapsed within 7 d after stopping the drug. Invalidation is that the clinical symptoms of the affected animals were not alleviated after treatment, and there was a tendency to increase.

Sample collection

At the end of the experiment, blood samples from 20 affected animals were collected from the jugular, placed in a heparin sodium vacuum tube for 60 min, centrifuged at 4 oC, 3500 r/min for 10 min, and stored at -80oC for testing.

Physiological and biochemical indicators

Hemoglobin (Hb), erythrocyte count (RBC), packed cell volume (PCV), corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and white blood cell count (WBC) were determined by automatic blood cell analyzer (SF-3000, Sysmex-Toa Medical Electronics, Kobe, Japan). Biochemical analyses, which included blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine transaminase (ALT), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), total antioxidant capacity (T-AOC), potassium (K), magnesium (Mn), and phosphorus (P) were determined by automatic biochemical analyzer (SF-1, Shanghai Medical Apparatus and Instruments Factory, Shanghai, China). Total protein (TP), albumin (ALB), globulin (GLB) were determined by electrophoresis (WH-300-LCD, Shanghai Shuangqi Biotechnology Co., Ltd., China).

Statistical analyses

Data were analyzed using the Statistical Package for the Social Sciences (SPSS, version 22.0, Inc., Chicago, IL, USA) and presented in the form of mean±standard error (SE). The differences were assessed by Student’s t-test and were extremely significant at P<0.01.

RESULTS

The incubation period is 1-2 d, mostly occurs in 6-week-old P. przewalskii. At the beginning, the body temperature of the juvenile P. przewalskii increased to above 41oC, conjunctiva became red and congested, and some of the affected juvenile P. przewalskii had neurological symptoms, stiffness of juncture, molars and dyskinesia. There are diarrhea symptoms. First, the yellow and white atheroma loose stool is discharged, and then the yellow and white watery loose stool is discharged. The undigested clot, exfoliated intestinal mucosa and blood silk are often mixed in the stool, and there are a lot of bubbles in the stool. Most of them died within 4-12 h after the onset of the disease, with a mortality rate of 80%. Affected P. przewalskii have abdominal pain symptoms, cannot stand, because of long-term diarrhea, and hindquarters were fecal pollution. Adult P. przewalskii have different symptoms, some of which are not obvious, some of which are mental depression, anorexia, diarrhea and almost no death. The pathological changes were mainly concentrated in gastrointestinal tract, presenting acute enteritis and systemic anemia. In the stomach, there are a lot of milk clots digested by the stomach, and the gastric mucosa is highly congested and edematous. The contents are in the form of blood and water. Some of the gastric mucosa falls off, the mesenteric lymph nodes are highly congested and swollen, the section is juicy, and the color of the kidney and liver is lighter. There are a lot of bleeding points under the renal capsule and the liver capsule, and the gallbladder is full. There is a large amount of hemorrhage in the endocardium. The whole blood is thin and cannot coagulate normally, and the spleen is significantly enlarged.

As shown in Figure 1, the cure rates of the low-dose and high-dose groups were 75% and 85%, respectively, and the cure rate of the gentamicin sulfate group was 60%. The effects of modified Yujin powder was better than gentamicin sulfate, however, the clinical symptoms of the P. przewalskii were still obvious.

 

As shown in Table II, the number of WBC in the low-dose group, the high-dose group, and the gentamicin sulfate group were extremely significantly lower (P<0.01) than those in the control group, but the difference between the experimental groups were not significant. There were no significant differences in other blood parameters between groups.

As shown in Table III, BUN and MDA contents in the low-dose group, the high-dose group, and the gentamicin

 

Table II. Hematological values of P. przewalskii.

Items	Low-dose group	High-dose group	Gentamicin sulfate group	Control group
Hb (g/L)	232.56±12.72	231.52±11.33	232.56±12.43	235.63±12.53
WBC (×109/L)	11.87±0.47B	14.69±0.66B	11.08±0.64B	28.02±3.65A
RBC (×1012/L)	12.88±0.63	12.82±0.55	13.73±0.74	13.76±0.88
PCV (%)	61.86±3.36	62.75±3.36	61.26±3.86	61.83±3.86
MCV (fL)	45.86±0.73	45.88±0.39	45.27±0.74	44.29±0.92
MCH (pg)	16.25±0.35	15.53±0.24	16.43±0.35	15.25±0.54
MCHC (g/L)	344.37±7.69	345.38±7.53	344.57±6.59	343.95±7.85

 

Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, corpuscular volume; PCV, packed cell volume; RBC, erythrocyte count; WBC, white blood cell count. Different capital letter superscripts indicate extremely significant differences at P<0.01 level.

 

Table III. Serum biochemical indices of P. przewalskii.

Items	Low-dose group	High-dose group	Gentamicin sulfate group	Control group
BUN (mmol/L)	1.79±0.33B	1.83±0.42B	1.87±0.34B	6.89±0.46A
AST (U/L)	89.87±8.47	89.75±8.32	89.08±8.64	88.52±8.65
ALT (U/L)	44.64±4.74	44.53±4.75	44.58±4.74	43.84±4.75
GSH-Px (U/mL)	114.64±21.35	113.64±21.36	113.64±21.85	112.64±21.86
SOD (U/mL)	94.43±9.24	95.75±9.74	94.73±9.62	94.84±7.63
CAT (U/mL)	16.64±1.43B	18.64±1.86A	16.63±1.24B	14.32±1.54C
MDA (nmol/L)	20.64±2.54B	24.53±2.32B	21.53±2.51B	33.64±2.25A
T-AOC (U/mL)	3.42±0.22A	4.95±0.32A	3.64±0.12A	2.52±0.21B
K (mmol/L)	3.51±0.22A	3.52±0.22A	3.55±0.20A	2.41±0.12B
Mn (mmol/L)	0.30±0.067	0.29±0.015	0.29±0.058	0.29±0.067
P (mmol/L)	2.85±0.43	2.83±0.34	2.84±0.25	2.82±0.54

 

BUN, blood urea nitrogen; AST, aspartate aminotransferase; ALT, alanine transaminase; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; T-AOC, total antioxidant capacity; K, potassium; Mn, magnesium; P, phosphorus. Different capital letter superscripts indicate extremely significant differences at P<0.01 level.

 

Table IV. Serum immunologic parameters of P. przewalskii.

Items	Low-dose group	High-dose group	Gentamicin sulfate group	Control group
TP (g/L)	56.76±5.78A	57.62±5.66A	56.53±4.64A	36.20±3.78B
ALB (g/L)	31.82±2.56A	31.52±3.65A	30.62±3.64A	23.78±3.99B
GLB (g/L)	24.67±2.71A	24.41±2.52A	23.51±2.53A	14.42±2.23B
A/G	1.29±0.16	1.29±0.25	1.30±0.19	1.64±0.23

 

TP, total protein; ALB, albumin; GLB, globulin; A/G, albumin/globulin. Different capital letter superscripts indicate extremely significant differences at P<0.01 level.

 

sulfate group were extremely significantly lower (P<0.01) than those in the control group, while K content and T-AOC level were greatly significantly higher (P<0.01) than those in the control group. There was no significant difference between the experimental groups. The activity of CAT in the high-dose group was extremely significantly higher (P<0.01) than that in the low-dose group, the gentamicin sulfate group, and the control group. The activity of CAT in the low-dose group and the gentamicin sulfate group was greatly significantly higher (P<0.01) than that in the control group. The difference between the low-dose group and the gentamicin sulfate group was not significant. There were no significant differences in other biochemical indicators between groups.

As shown in Table IV, TP, ALB, and GLB contents in the low-dose group, the high-dose group, and the gentamicin sulfate group were extremely significantly higher (P<0.01) than those in the control group, but the difference between the experimental groups was not significant.

DISCUSSION

Yang et al. (2004) study shows that Yujin has a protective effect on the gastrointestinal tract. Yujin also has the effect of relieving cold diarrhea, and compatibility with P. chinensis can reduce the adverse stimulation of the cold of the P. przewalskii (Lu et al., 2017). Shi et al. (2007) study shows that A. macrocephala has the effect of regulate qi-flowing for strengthening spleen, eliminating dampness and diuresis, and can improve the organism immune function. Shao et al. (2005) showed that C. vladimiria has an effect of promoting bile flow. C. vladimiria is warm in nature, and it has the effect of relieving pain and regulating the stagnation. It can be used in combination with A. macrocephala to treat “qi” deficiency of spleen and stomach, transport weakness, and vomite and eat less. Studies have shown that T. chebula and C. chinensis have anti-inflammatory effects (Cui et al., 2015; Li et al., 2015). Therefore, modern pharmacology believes that C. chinensis and T. chebula have a good inhibitory effect on E. coli, they have the functions of clearing heat and removing toxicity, and astringent. C. otophyllum can supplement blood, and G. uralensis can enhance palatability.

Animal blood physiological and biochemical indicators can not only reflect the physiological and health status of animals, but also can be used as one of the indicators to reflect the organism metabolic status (Chuku and Uwakwe, 2012; Saban, 2019; Allam et al., 2020; Fasae et al., 2015). The number of WBC in blood of the case greatly significantly increased, which may cause damage to the immune system of the organism. BUN is the final product of protein breakdown, which can more accurately reflect the balance between protein metabolism and amino acids in animals (Huo et al., 2020; Akhtar et al., 2020; Scott et al., 1982). In addition, the organism can maintain the balance of organism fluids through the kidneys and maintain the internal environment stability, and the changes in the organism fluids during abnormal renal function (Wang et al., 2014; Meng et al., 2019), such as vomiting, diarrhea, gastrointestinal drainage, can cause excessive potassium loss in the gastrointestinal tract. Modified Yujin powder can greatly significantly reduced BUN content and WBC, and extremely significantly increased K content. The levels of TP, ALB and GLB in serum can reflect the immune status of the animal’s organism (Ryabchenko et al., 2018). The results showed that modified Yujin powder can extremely significantly increased the contents of TP, ALB, and GLB. Therefore, modified Yujin powder can reduce the immune function of the P. przewalskii. The activities of GSH-Px, SOD, and CAT in serum reflect the organism antioxidant properties and ability to scavenge free radicals (Sinan et al., 2017; Song et al., 2020). Serum T-AOC level represents the total antioxidant capacity of the organism (Liu et al., 2019a, b). Serum MDA is a product of excessive oxidative free radicals attacking the rich unsaturated fatty acids in biofilms (Mahfuz et al., 2020; Iqra et al., 2019). It is a sensitive and reliable marker reflecting oxidative damage of tissues and cells caused by reactive oxygen species in organisms (Zeng et al., 2020; Allam et al., 2019). The study showed that the high-dose group can highly significantly increased the CAT activity, and was extremely significantly higher than the low-dose group and the gentamicin sulfate group. Compared with the control group, the high-dose group can extremely significantly reduced the MDA content, and greatly significantly increased the T-AOC. It indicated that the high-dose group can improve the scavenging ability of free radicals, reduce the damage of free radicals to cells, and effectively improve the antioxidant capacity of the P. przewalskii.

CONCLUSION

Modified Yujin powder had a good therapeutic effect on the E. coli in P. przewalskii, and the effect of the high-dose group was better. Modified Yujin powder can effectively enhance the immune and antioxidant functions of P. przewalskii, but the research on the active ingredients between Chinese medicine has yet to be further developed.

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (41671041), and the Research Fund for the Doctoral Program of Southwest University of Science and Technology (17zx7146).

Statement of conflict of interest

The authors have declared no conflict of interest.

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