| At present,the number of pet dogs in China is increasing,some common pathogens continue to infect dogs.Dogs are usually infected with Canine Respiratory Viruses(CRV)and Canine Enteric Viruses(CEV),making clinical diagnosis and treatment difficult.Therefore,assuming that rapid,accurate and multiple detection methods were established,valuable time will be spent on prevention and treatment of canine viral infectious diseases,which has important clinical significance.In this study,two modified Multiplex PCR methods(m PCRs)were established.Two sets of primers were designed and optimized for detection of CRV and CEV.The CRV included Canine Adenovirus Type 2(CAV2),H3N2 Subtype Canine Influenza Virus(CIV H3N2),Canine Distemper Virus(CDV),Canine Parainfluenza Virus(CPIV)and CEV included CAV2,Canine Circovirus(Canine CV),Canine Parvovirus(CPV)and Canine Coronavirus(CCo V).The sensitivity of the m PCR detection methods for CRV and CEV established in this study indicated that the minimum detection limit of m PCRs were both1×10~4 viral copies,and the specificity showed good results.When detected by different PCR instruments and at different times,the results were easy to be repeated.Twenty nasal swabs and 20 anal swabs were collected from dogs with respiratory or intestinal diseases for the clinical use of m PCRs.Results showed that 80%(16/20)of dog nasal swab samples were positive for CRV and CDV was the most common pathogen.85%(17/20)of dog anal swab samples were positive for the CEV and CPV was the most common pathogen,and there were several co-infectious cases.The established m PCRs can be used to investigate the status of CRV infection or CEV infection,providing an effective and accurate tool for rapid detection of common canine viruses.Among the CRV,CIV H3N2 has been a threat to dog health for more than a decade.The virus easily mutates and has the potential to spread across species.To investigate the genetic variation of CIV H3N2 in Guangdong,nasal swabs were taken from dogs with respiratory symptoms at animal hospitals or rescue centers in Guangzhou,Dongguan and Foshan in 2018.The m PCR test results showed that 16.27%(7/43)of the samples were positive for CIV.In order to optimize the isolation method of CIV H3N2,4‰of TPCK-trypsin was added to the CIV H3N2 positive swab samples,and that were inoculated in 9 days old chicken embryos.Finally,7 strains of CIV H3N2 were isolated,and the viruses with high hemagglutination titer was harvested within 48 hours,with a high success rate.7 strains of CIV H3N2 isolated from Guangdong province were analyzed by genetic evolution and key amino acid sites.The results showed that all the 7 strains were avian source CIV H3N2.The evolutionary relationship of the 6 strains was close to that of the previous Guangdong isolates,with no significant variation in the key sites.Surprisingly,one strain clustered in the same subbranch as the South Korean and American isolates,which was speculated to be related to South Korean or American strains,and this type of CIV H3N2 began to circulate in some areas of China.In summary,the current PCR detection methods of common viruses in dogs were improved in this study and the detection spectrum of the viruses was also expanded.Two new m PCR detection systems were developed,and two set of primer mixtures were used to detect a total of 7 viruses of dogs.The m PCRs have been proved to be specific,sensitive and stable,which greatly improved the efficiency of virus detection in clinical practice and facilitates epidemiological investigation in laboratory.Nasal swabs were collected from sick dogs in Guangdong province,and then m PCR tests were carried out.CIV H3N2 positive samples were used for virus isolation,identification and genetic evolution analysis.Finally,the genetic variation of CIV in Guangdong was mastered,which can provide theoretical basis for the diagnosis,treatment,monitoring and further prevention of CIV H3N2. |
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