Influencing Factors Of Subclinical Mastitis In Dairy Cows Based On DHI And The Establishment Of Multiplex PCR Detection Method For Mastitis Pathogens

Mastitis is a common disease in the breeding process of large-scale dairy farming.It can be classified into clinical mastitis and subclinical mastitis(hidden mastitis)based on the presence or absence of clinical symptoms.This disease often leads to a decline or loss of dairy cow’s production performance,causing significant economic losses to the livestock industry.The main pathogens that induce mastitis in dairy cows include Staphylococcus aureus,Klebsiella pneumoniae,Streptococcus agalactiae,Escherichia coli,and Salmonella enteritidis.In practical production,the identification and diagnosis of mastitis-causing pathogens mainly rely on observing lesions and changes in milk characteristics,combined with bacterial isolation and culture.However,these methods have drawbacks such as long processing time and low accuracy.Therefore,it is necessary to establish a multiplex PCR method capable of rapidly and accurately detecting multiple mastitis-causing pathogens simultaneously,to effectively carry out the prevention and control of mastitis in dairy cows.Dairy Herd Improvement(DHI),also known as dairy herd improvement,involves measuring milk production performance and milk composition of dairy cows and establishing a comprehensive recording system to assess the individual and genetic performance of cows and the herd.This provides guidance for dairy cow production management and genetic improvement.Some researchers have proposed using DHI data to systematically analyze the important factors influencing the occurrence of subclinical mastitis in dairy cows and determine the patterns of mastitis incidence in large-scale farms.This can contribute to the effective prevention and control of mastitis in dairy cows.This study first systematically analyzed the Dairy Herd Improvement(DHI)data from three large-scale farms in the northwest and east China regions to investigate the impact of factors such as season,parity,and lactation stage on the incidence of subclinical mastitis in dairy cows.Subsequently,by comparing the gene sequences of five common mastitis-causing pathogens in dairy cows(Staphylococcus aureus,Klebsiella pneumoniae,Streptococcus agalactiae,Escherichia coli,and Salmonella enteritidis),primers were designed for each pathogen based on their conserved genes to establish a multiplex PCR detection method.After program optimization,the specificity of the primers was validated using DNA from other mastitis-causing pathogens,and the sensitivity of the plasmid detection reaction containing conserved gene sequences was assessed.Finally,the multiplex PCR method was validated using mastitis milk samples collected from the farms.The experimental results are as follows:1.Analysis of the DHI data revealed the following results: the average incidence rates of subclinical mastitis in seasons A,B,and C were 15.4%,14.6%,14.0%,and 12.1% in summer,autumn,winter,and spring,respectively.There was a trend of higher incidence rates in summer and autumn compared to winter and spring,but the differences were not significant.Parity had a significant effect on the average incidence rate of subclinical mastitis in the three farms(P < 0.01),with the incidence rate gradually increasing with increasing parity.As lactation time increased,the average incidence rate of subclinical mastitis in different lactation stages showed a trend of initial decrease followed by an increase in the three farms,but there was no significant difference.The incidence rates of subclinical mastitis in the three farms were as follows: farm B had the highest rate(14.7%),followed by farm A(13.7%),and farm C had the lowest rate(12.3%).These results indicate that parity is the main factor affecting the incidence rate of subclinical mastitis in dairy cows,while the effects of season and lactation stage on the incidence rate are not significant.In the prevention and control of mastitis in dairy cows,it is necessary to focus on the impact of parity on mastitis incidence,take preventive measures in advance,and reduce the occurrence of mastitis.In addition,considering the regional differences in the incidence rates of subclinical mastitis in dairy cows on different farms,it is important to fully consider the effects of environmental factors such as geographical location and climate conditions on mastitis incidence rates in the daily management of farms and scientifically optimize feeding management programs.2.Through comparative analysis,primers were designed for the conserved genes of the five common mastitis-causing pathogens(nuc gene for Staphylococcus aureus,khe gene for Klebsiella pneumoniae,tuf gene for Streptococcus agalactiae,pho A gene for Escherichia coli,and inv A gene for Salmonella enteritidis).The expected sizes of the specific amplification products for Staphylococcus aureus,Klebsiella pneumoniae,Streptococcus agalactiae,Escherichia coli,and Salmonella enteritidis were 241 bp,414 bp,626 bp,740 bp,and 909 bp,respectively.After amplification and recovery of the specific bands for the corresponding genes of the five bacteria,five recombinant vectors including p MD 19T-nuc,p MD 19T-khe,p MD 19T-tuf,p MD 19T-pho A,and p MD 19T-inv A were successfully constructed for subsequent sequencing validation of the target fragments and sensitivity testing of the multiplex PCR method.The reaction conditions and program for the multiplex PCR were finally optimized as follows: primer working concentration of 0.8 μM,annealing temperature of 59.6℃,cycle number of 30,and extension time of 40 s.The multiplex PCR results showed that each positive sample had a single PCR amplification band,and there was no crossreactivity with other bacteria such as Streptococcus agalactiae,Enterococcus faecalis,Staphylococcus epidermidis,Staphylococcus saprophyticus,and Bacillus cereus,indicating good specificity.The results were consistent and showed good repeatability upon repeated validation.The sensitivity of the multiplex PCR method was tested using standard plasmidsimulated samples,and the detection sensitivity for all pathogens reached 5 copies/μL,indicating high sensitivity.Multiplex PCR was performed on 144 mastitis milk samples,and the results were as follows: 49 strains(34%)were infected with Staphylococcus aureus,31strains(21.5%)with Escherichia coli,14 strains(9.7%)with Streptococcus agalactiae,10strains(6.9%)with Klebsiella pneumoniae,3 strains(2.1%)with Salmonella enteritidis,33samples(23.6%)did not show infection by the aforementioned five pathogens,and 3 samples(2.1%)showed mixed infections.In conclusion,this study analyzed the impact of season,parity,and lactation stage on the incidence of subclinical mastitis in large-scale dairy farms based on DHI data.The study found that parity is the main factor influencing the incidence rate of subclinical mastitis.Additionally,the study successfully established a multiplex PCR detection method that can rapidly identify the five common mastitis-causing pathogens in dairy cows.This method has the advantages of high sensitivity,good specificity,and strong repeatability.The findings of this study provide a preliminary foundation for the prevention and control of mastitis in dairy cows and have potential for broader application in practice.