An Investigation Into The Pathogens Associated With Canine Infectious Respiratory Disease And Pathogenicity Analysis

Canine infectious respiratory disease(CIRD)is a constant challenge in dogs and is a highly contagious disease.This disease possesses a multifactorial aetiology,wherein multiple pathogens act sequentially or synergistically to cause the disease,which is common within large dog populations.In recent years,canine influenza virus(CIV)is a relatively new respiratory pathogen affecting dogs,while Staphylococcus pseudintermedius is another new emerging opportunistic pathogen in dogs.During influenza pandemics secondary bacterial infection is the main cause of the mortality.There is dire need to know mechanisms involved in the interaction between CIV and S.pseudintermedius to understand treatment procedures that fight viral and secondary bacterial infection.In the present study,pathogens associated with canine infectious respiratory disease were isolated,and molecular characterization and synergism between CIV H3N2 and S.pseudintermedius were investigated.1:Streptococcus pluranimalium isolated from a canine respiratory case:identification and experimental infection in miceS.pluranimalium was isolated firstly from a canine respiratory patient attending Veterinary Hospital,Nanjing Agricultural University,China.A 4-month-old Tactic dog was presented for evaluation and treatment of respiratory syndrome.One strain of Streptococcus pluranimalium was isolated and confirmed by biochemical characteristics and 16S rRNA sequence analysis.BALB/c mice were challenged by intraperitoneal injection with 108 CFU S.pluranimalium to determine the virulence of this bacterium.Lung,liver,heart,kidney,spleen and brain were harvested at intervals for analysis.Lung tissue showed the earliest bacterial detection at 2 days and all the collected tissues demonstrated greater colony counts at 96h h following exposure to the bacterium.Histopathological examinations demonstrated that lung cells became widened with thickening of the bronchiolar lumen and narrowing of alveolar septa.Exudate in macrophages resulted in significant increase in lung congestion and severe consolidation.The main lesions in the brain were characterized by edema of nerve cells and non-suppurative encephalitis,indicating that S.pluranimalium is capable of inducing brain damage by crossing the blood-brain barrier(BBB).It is concluded that S.pluranimalium may be one of causative agents for canine infectious respiratory disease.2:Isolation,identification and antimicrobial resistance of bacterial isolates from dogsMicrobiological and epidemiological studies were conducted for the examination of bacterial species,isolated from upper respiratory tract of dogs from Animal Clinics of Nanjing Agricultural University in Jiangsu Province of China from March 2013 to September 2013.Forty nasal swabs were taken from dogs having respiratory signs.The samples were cultured on culture media such as TSB,LB,blood agar and MacConkey's agar.S.pseudintermedius was the most frequently isolated pathogen(37.50%),followed by Staphylococcus aureus(18.75%),S.pluranimalium(10.93%),Streptococcus canis(9.37%),Staphylococcus schleiferi(9.37%),Staphylococcus intermedius(6.25%),Staphylococcus cohnii(4.71%)and Staphylococcus hominis(3.12%).The isolated organisms S.pseudintermedius and S.pluranimalium were subjected to commonly used antibiotics for antibiogram and determination of resistant drugs.S.pseudintermedius showed antimicrobial resistance to gentamicin(70.83%),tetracycline(50%)and oxacillin(45.83%),while S.pluranimalium had antimicrobial resistance to enrofloxacin(71.42%),gentamicin(57.14%)and trimethoprim-sulfamethoxazole(42.85%).3:Pathogenicity potential of S.pseudintermedius isolated from dogs with infection signsExperiments were performed to determine the pathogenicity characteristics of S.pseudintermedius isolated from dogs with infection signs in mice.The research was mainly focused on clinical symptoms,bacterial loads,histopathological changes and virulence factors.BALB/c mice were intranasaly inoculated with S.pseudintermedius.The results showed that the bacteria can cause serious neurologic symptoms including walking in circles and bending of neck.S.pseudintermedius infection produces obvious histopathological changes in lungs and brains of the mice.High levels of bacterial loads were observed in lungs and brain.Of all11 oxacillin-resistant isolates of S.pseudintermedius,2 carried SCCmec types?-?,2 carried SCCmec?and 3 carried SCCmec types?according to PCR-based SCCmec typing.Exfoliative toxin(siet),lukS and lukF genes were also identified from the isolates.4:Identification and genetic evolution analysis of one strain of H3N2 canine influenza virus isolated from Nanjing,ChinaNasopharyngeal swabs were collected from 6 dogs with severe respiratory syndrome at the Animal Clinics of Nanjing Agricultural University in 2014.One viral strain was isolated from SPF embryonated chicken eggs.Sequencing analysis of hemagglutinin(HA)and neuraminidase(NA)genes showed that the CIV isolate shared high similarity(above 98%)to the H3N2 viruses from dogs in China.After the comparison of gene sequence,four unique mutations were found in the amino acid of HA(A144T,R158K,D291N,L383F)and NA(T19A,V33L,V82A,S336N).The HA and NA genetic evolution analysis indicated that the CIV isolate was most similar to the newly isolated H3N2 viruses from dogs in China and they were in the same evolutionary branching.The result provided a foundation for further investigation of biological characteristics of CIV.5:Canine influenza virus coinfection with S.pseudintermedius enhances bacterial colonization,virus load and clinical presentationExperiments were conducted to investigate whether synergism exists between canine influenza virus and S.pseudintermedius(Sp).For this purpose,BALB/c mice were divided into various groups and were inoculated with CIV only,Sp only or both agents(CIV/Sp).The mice in the CIV/Sp co-infection group were challenged with S.pseudintermedius three days after CIV infection.During the experiments more severe clinical signs were observed in the CIV/Sp group,and the body weights of the mice in the co-infection group decreased significantly as compared to those of the mice in the CIV-only as well as Sp-only groups.CIV inoculation increased bacterial colonization,whereas secondary infection with S. pseudintermedius elevated the viral RNA titres of CIV in tissues.The histological studies showed that lesions in the brain,spleen and lung were more severe in the CIV/Sp group than in the singly treated groups.Infection in all groups i.e CIV alone,Sp alone or coinfection stimulated a significantly higher release of cytokines,such as IFN-y,IL-6,TNF-a and lymphotactin(Lptn),than was observed in the non-infected group.Moreover,the levels of IFN-y in the spleen and lung were higher in the CIV/Sp group compared to the CIV-only and Sp-only groups.Our findings provide the first demonstration that coinfection with CIV and S.pseudintermedius may synergistically worsen clinical signs and lesion.