Establishment And Application Of Common Pathogen Detection Methods On Feline Upper Respiratory Infections

In recent years,with the increase of the number of cat owners,the upper respiratory disease（FURD）of cats has been paid more and more attention because it is one of the important causes of morbidity and death of young cats.The typical clinical symptoms of FURD are fever,loss of appetite,depression of spirit,serous,mucous or purulent secretions in the eyes and nasal cavity,edema or ulcer in the oropharynx,salivation,and occasional coughing and sneezing.The common pathogens are feline calicivirus（FCV）,feline herpesvirus type 1（FHV-I）,mycoplasma felis（M.felis）,chlamydophila felis（C.felis）and bordetella bronchiseptica（Bb）.As the clinical symptoms caused by various pathogens are similar,it is difficult to judge the disease by clinical symptoms.The purpose of this study is to establish a multiplex PCR detection method for the pathogens of upper respiratory tract infection in cats:FCV,FHV-1,C.felis and M.felis,and to detect the pathogens of upper respiratory tract infection in pet cats in Guiyang area,so as to provide guidance for clinical treatment and reasonable prevention.1.Establishment of four-plex PCR detection methods for FCV,FHV-1,Mycoplasma felis and Chlamydophila felisThrough primer 5.0 software,four pairs of primers were designed to amplify the target gene fragments of FCV,FHV-1,M.felis and C.felis respectively,referring to the gene sequences of FCV ORF2（accession number:NC001481）,FHV-1 UL26（accession number:NC013590）,M.felis（accession number:NZ＿AP022325.1）and C.felis（accession number:AF269258）reported by NCBI.According to the positive swabs infected with the above four pathogens,nucleic acids were extracted as positive templates.By identifying the single PCR products of the four pathogens,and optimizing and testing their annealing temperature and sensitivity,a single-plex PCR method for rapid detection of FCV,FHV-1,M.felis and C.felis was established.On the basis of the established single PCR detection method,the annealing temperature,template and primer concentration were explored,and the specificity and sensitivity of four-plex PCR were tested in order to establish a multiplex PCR detection method for simultaneous and rapid detection of common pathogens of feline upper respiratory tract infection such as FCV,FHV-1,M.felis and C.felis.The results of the establishment of single-plex PCR detection method:（1）The best reaction system:the upstream and downstream primers were1μL,the template DNA/cDNA was 2μL,and the gold medal Mix（green）was25μL.（2）Optimum annealing temperature:the best annealing temperature range of single PCR amplification for four pathogens is 50℃～60℃.（3）Sensitivity results:the lowest detection concentrations of each pathogen gene were FCV 1×10^-5ng/μL,FHV-1 1×10^-6ng/μL,M.felis 1×10^-6ng/μL and C.felis 1×10^-5ng/μL.The results of the establishment of four-plex PCR detection method（1）The best reaction system:the four pairs of upstream and downstream primers are 1μL each,the cDNA template of FCV is 1μL,the DNA of FHV-1,mycoplasma and chlamydia is 1μL each,and the gold medal Mix（green）makes up 50μL.（2）Optimum annealing temperature:the optimum annealing temperature range of the reaction system is 51.7℃～60℃,in which the band is the clearest at 60℃.（3）Sensitivity test:the minimum detection concentration of FCV gene was 1×10^-5ng/μL,FHV-1 gene was 1×10^-4ng/μL,M.felis target gene was1×10^-3ng/μL,C.felis target gene was 1×10^-4ng/μL.（4）Specificity test:The PCR reaction tube was added to the reaction system of DNA extract from pathogenic swabs with FCV,FHV-1,M.felis,C.felis,healthy cat swabs,swabs with feline parvovirus（FPV）and（MB）swabs from bovine mycoplasma.The target gene fragments of FCV,FHV-1,M.felis and C.felis were amplified respectively,but no other gene fragments were amplified.2.Application of four-plex PCR for detection of FCV,FHV-1,Mycoplasma felis and Chlamydophila felisUsing the established four-plex PCR detection method for four common pathogens of feline upper respiratory tract infection,a total of 170 samples of ocular conjunctiva,nasal mucosa and oropharyngeal mucosa swabs of cats with upper respiratory tract infection were detected in an animal hospital in Guiyang from October 2019 to September 2020.The test results showed that among the 170 clinical samples,65 samples detected the target gene,and the detection rate was 38.32%（65/170）,including 34 cases of single pathogen infection（20%）and 31 cases of multiple pathogen infection（18.23%）.The target gene was not detected in 102 samples.A total of 33 FCV positive samples were positive,with a positive rate of 19.41%（33/170）;29 FHV-1positive samples,with a positive rate of 17.05%（29/170）;34 M.felis positive samples,with a positive rate of 20%（34/170）;and 1 C.felis positive sample,with a positive rate of 0.5%（1/170）.The established four-plex PCR method for FCV,FHV-1,Mycoplasma felis and Chlamydia felis can be applied to the detection of corresponding clinical samples,and provide reference for the epidemiological investigation of pathogens and the subsequent five-plex PCR method for detection of FCV,FHV-1,Mycoplasma felis,Chlamydia felis and Bornella bronchosepticus.