| The distribution and characteristics of bovine pathogenic E. coli were studied by the isolation of E. coli which was from fecal samples originated in the rectums of the dairy cows, animal pathogenicity test, biochemical characteristics test, serotygrouping and multiplex PCR test. And the antibacterial activities of the herbal medicines on bovine E. coli with different serogroups were also discussed. Meanwhile, the inhibitory effect of calcium cyanamide added as antibacterial agent in cattle manure composting on bovine pathogenic E. coli with different serogroups were tested. The research will provide an effective prevention and control measure for E. coli spread and infection, and it is meaningful to the healthy development of China's dairy industry and combating disease outbreaks. And the application of calcium cyanamide on composting proposed a new feasible route to its use. The content of the experiment included three portions as follows.1. Isolation and identification of bovine pathogenic E. coli, detection of E. coli virulence factor and medicine testIsolation of E. coli, biochemical characteristics test, serotygrouping and multiplex PCR test were carried out to isolate and identify bovine pathogenic E. coli originated in fecal samples which were collected from 206 and 212 dairy cows in Baoto City and Hulunbeier City of Inner Mongolia, respectively. Then, the antibacterial activities of the herbal medicines on these bovine E. coli with different serogroups were studied. The results showed:⑴E. coli was isolated and identified from 83.50% fecal samples collected in Baoto City, and of which 40.12% belonged to pathogenic E. coli. The dominant serogroups of the pathogenic E. coli were O78, O2, and O8, accounting respectively for 38.89%, 33.33% and 14.81% in all E. coli isolates. E. coli was isolated and identified from 47.64% fecal samples collected in Hulunbeier City, and its dominant serogroups were O149, O131, O2 and O96,accounting respectively for 9.30%,8.14%,6.98% and 6.98% in all E. coli isolates.⑵33.66% fecal samples collected and 27.72% E. coli strains isolated from Hulunbeier City carried E. coli virulence factors. Among the virulence factors, eaeA was with the highest detection rate, which was found in 26.73% fecal samples and 22.11% E. coli strain. And atypical pathogenic E. coli (aEPEC) was with widest distrubution, which was detected from 21.78% fecal samples and 18.15% E. coli strain.⑶The antibacterial activities of the herbal medicines on E. coli strains from Hulunbeier City in order were Rhzoma Atractylodis Lanceae, Fructrs trichosanthis, Radix Scutellariae Baicalensis, Rhizoma Coptidis, and Taraxaci mongolicum. The antibacterial activities on E. coli strains from Baoto City in order were Radix Scutellariae Baicalensis, Rhizoma Coptidis, Fructrs trichosanthis, Rhzoma Atractylodis Lanceae, and Taraxaci mongolicum. The result indicated that antibacterial activity of different drugs varied in vitro and the antibacterial activity of same drug on bovine pathogenic E. coli strains with different serogroups were also different.2. Inhibitory effect of calcium cyanamide on bovine pathogenic E. coli with different serogroups in fresh cow manure compostingThrough laboratory scale composting, the test researched on the inhibitory effect of calcium cyanamide on the bovine pathogenic E. coli with different serogroups by adding 0%, 2%, 3% and 4% calcium cyanamide for fresh cow manure composting to culture under 20℃, 30℃, 37℃, 50℃and 60℃conditions, and also discussed its inhibitory effect on endogenous E. coli, mesophilic bacteria and thermophilic bacteria of compost. Then, moisture content of the compost on the antibacterial effects of calcium cyanamide was tested through adjusting the moisture content of sterilized manure with sterile water (respectively adjusted to 45%, 65% and 85%) after adding 0%, 2%, 3% and 4% calcium cyanamide for composting. In addition, the direct effect of calcium cyanamide on the growth of bovine pathogenic E. coli was analyzed by culturing the medium with different concentrations of calcium cyanamide (0.01-10.00 mg·mL-1) inoculated with E. coli inoculum with different serotygroups. The results showed:⑴Fresh cow manure compost added by 3% and 4% calcium cyanamide killed the pathogenic E. coli with O2 and O8 serotygroups either 50℃and 60℃high temperature or 20℃, 30℃and 37℃low temperature conditions. The antibacterial effect of calcium cyanamide under high temperature conditions was better than under low temperature conditions. And the inhibitory effects of calcium cyanamide on E. coli strains with O2 and O8 serogroups were significantly different, its effect on endogenous E. coli of compost also showed a similar pattern.⑵Fresh cow manure compost added by 3% calcium cyanamide had a certain influence on the growth of mesophilic and thermophilic bacteria under 30℃and 50℃conditions, however, mesophilic and thermophilic bacteria still remained normal growth pattern.⑶The inhibitory effect of calcium cyanamide on bovine pathogenic E. coli was closely related to the moisture content of compost. The compost with 85% moisture content played the most beneficial promoting influence on antimicrobial effect of calcium cyanamide, followed by 65%, and the weakest is 45%, indicating that compos with high moisture content is helpful to inhibitory effect of calcium cyanamide.⑷Calcium cyanamide presented the inhibitory effect on the growth of bovine pathogenic E. coli with different serogroups. With increasing the concentration, the inhibitory effect of calcium cyanamide on the bovine pathogenic E. coli was significantly enhanced. Moreover, the inhibitory effect of calcium cyanamide was affected by the temperature, 50℃and 60℃temperatures played a beneficial role in it. Besides, the inhibitory effect of calcium cyanamide on the growth of bovine pathogenic E. coli with different serogroups was different.3. The effect of calcium cyanamide on composting progressThe raw materials of the composting added 0%, 2% and 3% calcium cyanamide were inoculated with bovine pathogenic E. coli inoculum with different serogroups (O2, O78 and O86) , then composted by 63 days time. During the composting time, the physical, chemical and biological parameters were regularly determined. The results showed:⑴For the compost added 2% calcium cyanamide, Compost temperature reached the maximum 40.1-45.9℃in 29-31d, pH value reached 8-9 in 35d. Starting from the 42d, T value was down to 0.72. At the end of composting, GI reached 140.16-155.70%. For the compost added 3% calcium cyanamide, there was no high temperature period, the temperature ranged from 19.6℃to 28.7℃, pH value was constantly higher than 9. At the end of composting, T value was still high, and GI reached 119.87-129.55%.⑵The composts added 2% and 3% calcium cyanamide had no significant impact on eutrophic bacteria, oligotrophic bacteria and actinomycetes, however, have a certain influence on proteolytic bacteria when the compost added 2% calcium cyanamide affected the activity of proteolytic bacteria. And they were no significant impact on amylolytic and cellulolytic bacteria, while promoting the activity of lipolytic bacteria.⑶The composts added 2% and 3% calcium cyanamide significantly shortened the time for killing E. coli in compost. With increasing the addition doses, inhibitory effect of calcium cyanamide was significantly improved.In short, the fresh cow manure compost can effectively kill the bovine pathogenic E. coli with different serogroups by adding calcium cyanamide. Meanwhile, the addition of 2% calcium cyanamide on compost does not influence the composting progress. Thus, through adding calcium cyanamide on compost, we can not only achieve the purpose of safe disposal for manure, but also accomplish the recycle utilization of resources. |
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