| Calf diarrhea seriously harms China’s dairy farming industry,with high mortality rate,large economic losses,complicated causes,and lack of effective prevention and control technologies.From 2018 to 2020 year,the team investigated 65715 calves from 78 large-scale dairy farms in Tangshan,the average incidence rate of diarrhea was about 11.96%,and the mortality rate was about 40.37%,which seriously affected the development of Tangshan City dairy industry.The purpose of this study is to clarify the main infectious pathogens of calf diarrhea in Tangshan area and improve the diarrhea prevention and control capabilities of dairy farms.Use laboratory pathogen detection methods to conduct epidemiological investigations on 9 main infectious diarrhea pathogens,clarify the main pathogens of calf diarrhea,reveal the current status of calf infectious diarrhea pathogens and their distribution in dairy farms;establish major Diagnosis and detection methods of viral pathogens;development of compound Chinese medicine preparations for the treatment of diarrhea,providing data support for the prevention and treatment of the main pathogens of calf diarrhea.In this study,9 major pathogens were tested for calf diarrhea feces in 13 counties(cities,districts)in Tangshan area.Statistics showed that among 788 diarrhea samples,the positive detection rate of Bovine viral diarrhea virus was 29.82%(235/788),the positive detection rate of Bovine rotavirus was 29.44%(232/788),and the positive detection rate of Bovine rotavirus was 18.02%(142/788);the positive detection rate of Escherichia coli was 43.53%(343/788),the positive detection rate of Salmonella was 22.46%(177/788),and the positive detection rate of Clostridium perfringens was 11.55%(91/788);the positive detection rate of Cryptosporidium was 27.79%(219/788),the positive detection rate of Coccidia was 27.16%(214/788),and the positive detection rate of Giardia was 9.77%(77/788).From the perspective of mixed infections,it was found that in 788 samples,mixed infections of viruses,bacteria,and intestinal protozoa accounted for 21.19%(167/788)of the total number of samples.In the double mixed infections,Bovine rotavirus+Bovine coronavirus,Escherichia coli+Salmonella,coccidia+Cryptosporidium,the positive detection rates were 8.37%(66/788),6.47%(51/788),5.20%(41/788);the triple mixed infection combination was Bovine virus diarrhea virus+Cryptosporidium+Escherichia coli 3.93%(31/788),quadruple mixed infection of Bovine viral diarrhea virus+ Bovine coronavirus+Escherichia coli+Salmonella is the highest,which is 0.89%(7/788),no mixed infections of five or more were detected.The survey results showed that among the pathogens of calf diarrhea within the sampling range,Escherichia coli accounted for the highest proportion,followed by Bovine viral diarrhea virus,Bovine rotavirus.The pathogens of calf diarrhea in Tangshan area are mainly concentrated in large-scale breeding farms in Luannan County,Luanzhou City and Yutian County,which has a certain relationship with the number and density of breeding.Among all inspected areas,Luannan County has the highest infection rate of Bovine viral diarrhea virus,with a positive detection rate of 45.38%(59/130),and Hangu District has the highest infection rate of Bovine rotavirus and Bovine coronavirus,positive detection rates were 55.00%(22/40)and 37.50%(15/40)respectively;the Guye District had the highest Escherichia coli infection rate,with a positive detection rate of 68.75%(11/16),and Salmonella infection rate in Lunan District The highest positive detection rate was 35.89%(14/39),and the positive detection rate of Clostridium perfringens in Qian’an County was the highest,20.21%(19/94);the infection of Cryptosporidium and Coccidia in Lubei District The rate of positive detection was 48.48%(16/33)and 57.57%(19/33)respectively.The infection rate of Giardia in Guye District was the highest,with a positive detection rate of 18.75%(3/16).The survey results show that there are three types of pathogen infections in dairy farms in different regions,and the pathogen detection rate is very different.Diarrhea occurred all year round.The highest infection rate of Bovine rotavirus was 27.71%(46/166)and 39.58%(114/288)in spring and summer,and the main infection rate of Bovine viral diarrhea virus was 52.17%(72/138)in winter.Bacterial and intestinal protozoa infection occurred frequently in autumn,and the positive detection rates of Escherichia coli,Salmonella and Clostridium perfringens were 54.08%(106/196),29.59%(58/196),14.58%(28/196).The positive rates of Cryptosporidium,coccidia and Giardia were 46.94%(92/196),41.84%(82/196)and 17.35%(34/196),respectively.The E2 gene of Bovine viral diarrhea virus was systematically analyzed by bioinformatics.The sequences with higher antigenicity and hydrophilicity were intercepted and synthesized and ligated to the prokaryotic expression vector pET-32a(+)to construct the recombinant plasmid pET32a-E2;transformed into E.coli BL21(DE3)host strain,analyzed by SDS-PAGE and Western blot,successfully obtained the best soluble protein of 39 kDa Bovine viral diarrhea virus E2 gene,and the protein has strong specificity;The ELISA antibody detection method established by the recombinant protein pET32a-E2 with good reactogenicity has the best detection effect when the enzyme-labeled antibody is diluted 1:10000;the coefficient of variation of the repeated experiments within and between groups of this method is less than 10%;The specific test results show that there is no antigenic cross-reaction with the positive serum of Bovine rotavirus,Bovine coronavirus and Bovine infectious rhinotracheitis virus.A multiple fluorescent quantitative PCR method for simultaneous detection of Bovine viral diarrhea virus,Bovine rotavirus and Bovine coronavirus has been established.The results showed that the minimum detection limits for the plasmid standards of Bovine viral diarrhea virus,Bovine rotavirus and Bovine coronavirus were 1.19×102 copies/μL,3.89×101 copies/μL,3.74×101 copies/μL,the lowest sensitivity is 100 times higher than conventional PCR,with high sensitivity;This method only specifically amplifies Bovine viral diarrhea virus,Bovine rotavirus and Bovine coronavirus.and does not cross-react with Escherichia coli,Salmonella,and Bovine infectious rhinotracheitis virus,and has strong specificity;The coefficient of variation within the group is less than 1%,and the coefficient of variation between groups is less than 1%,which has good repeatability;the established method and conventional multiplex PCR are used to simultaneously detect 150 samples of diarrheal disease materials collected in clinic,and the mixed infection of three viruses is detected at the same time The coincidence rate of the two samples is 83.33%.The results show that the multiple fluorescent quantitative PCR detection method established in this experiment provides a rapid,sensitive and specific technical means for the clinical diagnosis and epidemiological monitoring of Bovine viral diarrhea virus,Bovine rotavirus and Bovine coronavirus.Based on the principles of clearing heat and removing dampness,regulating qi and relieving pain,and stopping diarrhea,compound Chinese medicine preparations were developed.To study the acute toxicity of this prescription on the LD50 and maximum dose of mice.When the dose of the compound Chinese medicine preparation was increased to 21.50 g/kg,the mice did not die,and the LD50 of the compound Chinese medicine preparation could not be measured;During the maximum dose test,the clinical performance of the mice was normal,no poisoning occurred,no death was found,and the feed intake and body weight of the mice were not significantly affected(P>0.05).The maximum dose of the compound Chinese medicine preparation 120 g/kg;hematology and blood biochemical indexes showed no significant difference(P>0.05);No pathological changes were found in the heart,liver,kidney and other organs during autopsy,and the coefficient of each organ was not significantly different between the two groups(P>0.05);it indicated that the compound Chinese medicine preparation had no toxic side effects on the body of mice.To study the therapeutic effect of compound traditional Chinese medicine preparations on calves naturally infected with viral diarrhea.A single-factor design experiment was used to divide the calves into 3 groups(compound Chinese medicine treatment group,ribavirin treatment group,and compound Chinese medicine+ribavirin treatment group).In addition,healthy calves without diarrhea symptoms were selected as the control group.Each of these groups was 30 calves.the trial period was 15 days,On days 0,3,7,and 14 of the experiment,calves were weighed and blood samples were collected for blood indexes.The combination of several drugs in the compound Chinese medicine prescription can effectively improve the clinical diarrhea symptoms of sick cattle,with a relative weight gain of 80.89%and a cure rate of 100.00%.Its effect is better than that of a single ribavirin.This study through the etiology and epidemiology of calf diarrhea in Tangshan area,has a more comprehensive understanding of the prevalence of calf diarrhea and related pathogens in Tangshan area,and established the main pathogens of calf diarrhea.The method of diagnosis and detection,the development of a compound Chinese medicinal preparation for the treatment of calf diarrhea,and achieved good application effects.The results of this study will provide data reference for the prevention and treatment of calf diarrhea in Tangshan area. |
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