Z. Qunhui,1 Ch. Mingyong,2 Zh. Bing,2 S. Qinye2 and W. Jiemei2
1. Department of Animal Science, Agriculture and Animal Husbandry College, Nyingchi 860000, Tibet,
2. College of Veterinary Medicine, China Agriculture University, Beijing 100094, P.R. China
Three strains of enterotoxigenic E. coli isolated from dead yak were cultured in improved Mundell medium and the resulting filtrates were collected. The ileum ligation test was done with adult rabbits and mice aged 1–3 days by infusing the filtrates. The results indicate that three strains of enterotoxigenic E. coli produce toxic agents. This may be the cause of diarrhoea and subsequent death of yak.
Keywords: Enterotoxicity, enterotoxigenic E. coli (ETEC), yak
Pathogenic E. coli producing toxic agents are generally called enterotoxigenic E. coli (ETEC). ETEC can produce an exotoxin called enterotoxin, which mainly affects intestinal wall. There are two kinds of enterotoxins so classified on the basis of stability to heat: heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST), including STI and STII. The present study involved investigations of diarrhoea epidemics in Naqu yak in Tibet, P.R. China. Pathogen identification revealed that pathogenic E. coli was the causal organism. Test of enterotoxicity was done in order to explore the pathogenic reasons.
Three strains of enterotoxigenic E. coli (9901, 9903 and 9904) were isolated from Tibetan yak, which died during an epidemic.
The standard positive strain was obtained from the Supervision Centre for Veterinary Medicine in China.
Adult rabbits and newborn mice aged 1–3 days were obtained from the Institute for Experimental Animal of Chinese Academy of Medicine.
Improved Mundell medium was used to culture the strains. The culture was inoculated for 18 hours at 38°C. Shaking was required in the middle of the culture and the liquid culture was turbid.
The liquid culture was centrifuged for 15 minutes at a speed of 10,000 rpm and a film with diameter of 0.22 μm then filtered the supernatant. The filtrate was stored at 4°C for later use.
Heat-labile enterotoxin (LT): Healthy rabbits weighing 2–3 kg, fasted for 36–48 hours, were used to do the ileum ligation test. Five segments were ligated, of which two segments were used as positive and negative controls each with infusion of 1 mL positive filtrate from the standard strain and 1 mL culture medium as negative, respectively, and the other four segments were infused with 1 mL of the filtrates from the strains of 9901, 9903 and 9904, respectively. Following 18–24 hours fasting, the rabbits were sacrificed and the ligated segments from the ileum were removed. The amount of liquid retained in each ligated segment was measured, and those, which had more than 1 mL liquid, were considered as positive. The length of each ligated segment was also recorded.
Heat-stable enterotoxin I (STI): Nine newborn mice aged 1–3 days were used to detect the STI in the filtrates of strains 9901, 9903 and 9904 by stomach lysis test. The animals were divided into five groups randomly. Each group had 2 mice except one, which had only 1 used as control. The filtrates were injected in the 1st, 2nd and 3rd groups, respectively. The positive filtrate of the standard strain and the culture medium were infused into the 4th and 5th groups as positive and negative controls, respectively. Each young mouse was injected with 0.1 mL of the liquids and kept alive for 2–4 hours at 25°C. The intestine and the remains of each mouse were weighed after the mouse was sacrificed. The ratio (G/C) of intestine [G] to the remains [C] was considered positive if it was more than 0.083.
Heat-stable enterotoxin II (STII): Adult mice were used to detect the STII in the filtrates of strains 9901, 9903 and 9904 by the ileum ligation (four segments of each animal) similar to that for the rabbits mentioned above. Of the collected filtrates 0.2 mL were infused into the two segments, and 0.2 mL positive filtrate of the standard strain and 0.2 mL culture medium were infused into the other two segments each as controls, respectively. Ratio (W/L) of the liquid retention in the ligated segments (mg) to the length of the ligated segments (cm) was considered as positives for the enterotoxigenic STII if it was more than 10.
In the detection of the filtrate of strain 9901, the liquid retentions in the four ligated segments were 1.00 mL, 1.27 mL, 1.10 mL and 1.15 mL, respectively. The positive control was 1.15 mL and the negative one 0.70 mL. Corresponding results for strain 9903 were 1.51 mL, 2.75 mL, 2.36 mL and 1.25 mL, respectively, and the positive and negative controls were 2.48 mL and 0.95 mL, respectively. For strain 9904, the results were 1.00 mL, 1.29 mL, 1.43 mL and 1.25 mL, respectively, and corresponding positive and negative controls were 1.50 mL and 0.80 mL, respectively. Hyperemia of mucosa blood capillary was observed on the surface of experimental ileum of 9901 and 9903. They also had hemoid mucus. The positive control had the same features, but the negative one was different. Strain 9904 had the same appearance and the liquid in it was aqueous. The results indicate that the three strains of E. coli produce LT (heat-labile enterotoxin).
The G/C ratios for strain 9901 were 0.191 and 0.171. Corresponding values for strain 9903 were 0.218 and 0.210 while those for strain 9904 were 0.218 and 0.215. The ratios of G/C for the positive control were 0.238 and 0.153 while the negative one was 0.055. These show that the three strains of E. coli produced STI.
The ratios of W/L for strain 9901 were 40.5 and 50.2, while values for the positive and negative were 79.2 and 8.56, respectively. Mucosa of the experimental segments and the positive segment were hyperemic after the ileum was clipped. However, the ratios of W/L for strain 9903 were 137.3 and 148.5, and those for the positive and negative controls were 139.3 and 8.72, respectively. The ligated segments were full of hemoid mucus and the ileum mucosa was bleeding and had substantial hemoid seepages. The W/L ratios for strain 9904 were 151.1 and 146.1, and the corresponding values for the positive and negative controls were 172.2 and 6.7. Dark brown hemoid mucus was found in the positive and the experimental segments. The seepage in the negative segment was aqueous. The results suggest that the three strains produce STII, and that strain 9903 was hemoid, 9904 was aqueous and 9901 was intermediate.
In conclusion, all the tests in this study show that the three strains of 9901, 9903 and 9904 produced enterotoxins and that these are probably responsible for the diarrhoea in yak.
The authors are grateful to Professor Chen Dewei for his advice and assistance in this work