Investigation On Infection Of Tuberculosis And Brucellosis In Dairy Cows In Guizhou Province And Establishment Of Milk Safety Evaluation System

Bovine tuberculosis(TB)and brucellosis in dairy cows seriously threaten the development of cattle industry and human public health security.In 2012,the General Office of the State Council issued the “National Medium and Long-Term Plan for the Prevention and Control of Animal Diseases(2012-2020)”,In 2012,the General Office of the State Council issued the National Medium-and Long-term Plan for the Prevention and Control of Animal Epidemics(2012-2020),which classified tuberculosis and brucellosis in dairy cows as two kinds of animal epidemics,and formulated comprehensive prevention and control measures for them.This study which prescribed dairy tuberculosis and brucellosis investigation programs is based on the "2017 National Animal Disease Surveillance and Epidemiology Survey Plan(Agricultural Medicine [2017] No.7)","National Animal Disease Special Epidemiological Survey Implementation Plan(Agricultural Medicine Defense Letter [2016] No.322))","Guidelines for the Prevention and Control of Tuberculosis in National Dairy Cows(2017-2020)(Agricultural Medicine [2017] No.20)" and "National Brucellosis Control Plan(2016-2020)"(Agricultural Medicine [2016] No.38)).In addition,the study investigated the infections of tuberculosis and brucellosis in dairy cows in Guizhou,and the infections of tuberculosis and brucellosis in dairy cows in Guizhou was preliminarily well known.Then,a rapid,accurate and reliable duplex PCR diagnostic method for bovine tuberculosis and brucellosis was established.Finally,this study identified the safety evaluation system testing items and indicators,and established an implementable safety evaluation system based on the research and analysis of milk nutrition safety and health safety,and the reference to relevant national standards and regulations,so as hopefully to provide a reference for the dairy cows Farming and milk safety protection in Guizhou.1 Investigation on tuberculosis and brucellosis infection in dairy cows in Guizhou ProvinceIn order to understand the prevalence of tuberculosis in dairy cows in Guizhou Province,a total of 106 large-scale dairy farms in 9 cities of Guizhou Province were randomly selected from 2017 to 2018.During the two years,20 478 samples of anticoagulant fluid were collected for bovine tuberculosis IFN-gamma ELISA test.Ten positive samples were detected and the individual positive rate was 0.05%(10/20 478),7 of which were detected in 2017,and the individual positive rate was 0.07%(7/10 306).While the other 3 positive samples were detected in 2018,and the individual positive rate was 0.03%(3/10 172).It is clear that the positive rate decreased in 2018 compared with 2017.In order to investigate the infection of brucellosis,27 435 whole blood samples of 106 cows from 9 cities in the province were randomly collected during the 2017-2018 trial.Among them,6 positive samples were detected,the positive rate was 0.02%(6/27 435),and the same field group was positively detected continuously in 2017-2018.In 2017,there were 4 positive populations,and in 2018,it decreased to 2.The survey results show that in recent years,the prevention,control and epidemic prevention measures for tuberculosis and brucellosis in dairy cows in Guizhou Province have had certain achievements,and the infection rate of the two infectious diseases has shown a downward trend.2 Establishment and application of double PCR diagnosis method for tuberculosis and brucellosis in dairy cowsReferring to the gene sequences of Mycobacterium tuberculosis pnc A(AF212297)and Brucella BM 21(AL27674)in NCBI,a pair of specific primers were designed for the amplification of Mycobacterium tuberculosis.The partial sequence of the pnc A gene and the Brucella BM 21 gene was 416 bp and 282 bp in size.By using the positive material provided by the cooperative unit,the annealing temperature and sensitivity of the single PCR of the two bacteria were optimized and tested,and the optimal annealing temperatures were 57 °C of Mycobacterium tuberculosis and 59 °C of Brucella.Then the minimum detectable amounts from sensitivity were 68 pg of M.bovis and 43 pg of Brucella.Secondly,the appropriate double PCR buffer system and enzyme were selected to optimize the annealing temperature,template and primer concentration.The optimal reaction system was 25 ?L,and the optimal annealing temperature was 59 °C.Finally,the specificity of the duplex PCR was explored.Sensitivity,Results The minimum detection of the two bacteria in the duplex PCR was 68 pg of M.bovis and 43 pg of Brucella,respectively.It proved that the duplex PCR assay established by the test has high sensitivity and specificity.Characteristics,this test successfully established a duplex PCR detection method that can simultaneously and quickly detect bovine tuberculosis and brucellosis.246 cases of bovine tuberculosis and brucellosis were detected in some areas of Guizhou Province from 2017 to April 2019 by the established double-PCR method.The results of double-PCR showed that one of the 246 cases was positive for bovine tuberculosis,the infection rate was 0.4%(1/246);two cases were positive for brucellosis,the infection rate was 0.81%(2/246);one case was mixed with bovine tuberculosis and brucellosis,and the infection rate was 0.81%(2/246).The coinfection rate was 0.4%(1/246),and the coincidence rate of double and single PCR was 100%.As a result,there are mixed infections of tuberculosis and brucellosis in cattle in Guizhou Province.The duplex PCR method established in this study is of great significance for rapid diagnosis of single or mixed infections in dairy farms.3 Establishment and promotion of milk safety evaluation systemIn order to ensure the safety of milk,through the investigation of dairy cattle breeding in Guizhou Province,the analysis and discussion of the nutritional and health safety of milk,the safety evaluation system of this experiment was determined to detect whether there were Mycobacterium tuberculosis and Brucella bovis in milk,whether the milk protein content was up to the standard,and whether lead,mercury,sulfonamides,chloramphenicol,melamine and aflatoxin in milk exceed the standard.Besides,in order to the relevant national and international standards and regulations,the safety evaluation system determined the indicators of the items as followed: no detection of Mycobacterium bovis and Brucella;no detection of chloramphenicol and melamine;no less than 2.8 g/100 g lactoprotein,no more than 0.05 mg/kg lead,no more than 0.01 mg/kg mercury,no more than 0.10 mg/kg sulfonamide residues,yellow.Aspergillus M1 should not exceed 0.5 ug/kg,and an implementable evaluation system should be established.In this scheme,firstly,the established duplex PCR method was used to detect bovine tuberculosis and brucellosis,and then the follow-up items were detected.In this scheme,firstly,the established duplex PCR method was used to detect bovine tuberculosis and brucellosis,and so would be used in the following detection.In order to verify the feasibility and practicability of the scheme,2 123 milk samples were collected from different farms in Guizhou Province from 2017 to April 2019.These samples were tested for bovine tuberculosis and brucellosis,as well as heavy metal lead,mercury content,sulfonamide residues,melamine,aflatoxin M1,chloramphenicol content,etc.No lead,chloramphenicol,melamine unqualified milk samples were detected,4 bovine tuberculosis and brucellosis unqualified samples were detected,17 milk protein content unqualified,26 sulfonamide content exceeded the standard,11 aflatoxin M1 exceeded the standard.According to the statistical analysis of the test data,the recommended indicators for milk safety in Guizhou were obtained.The results show that the safety evaluation scheme can be popularized and implemented.