Canine infectious respiratory disease (CIRD) is a constant challenge in dogs and is a highlycontagious disease.This disease possesses a multifactorial aetiology, wherein multiplepathogens act sequentially or synergistically to cause the disease, which is common withinlarge dog populations.In recent years, canine influenza virus (CIV) is a relatively newrespiratory pathogen affecting dogs, while Staphylococcus pseudintermedius is another newemerging opportunistic pathogen in dogs.During influenza pandemics secondary bacterialinfection is the main cause of the mortality.There is dire need to know mechanisms involvedin the interaction between CIV and S.pseudintermedius to understand treatment proceduresthat fight viral and secondary bacterial infection.In the present study, pathogens associatedwith canine infectious respiratory disease were isolated, and molecular characterization andsynergism between CIV H3N2 and S.pseudintermedius were investigated.
　　1: Streptococcuspluranimalium isolated from a canine respiratory case: identification and
　　experimental infection in mice
　　S.pluranimalium was isolated firstly from a canine respiratory patient attending VeterinaryHospital, Nanjing Agricultural University, China.A 4-month-old Tactic dog was presented forevaluation and treatment of respiratory syndrome.One strain of Streptococcus pluranimaliumwas isolated and confirmed by biochemical characteristics and 16S rRNA sequence analysis.BALB/c mice were challenged by intraperitoneal injection with 108 CFU S.pluranimalium todetermine the virulence of this bacterium.Lung, liver, heart, kidney, spleen and brain wereharvested at intervals for analysis.Lung tissue showed the earliest bacterial detection at 2 daysand all the collected tissues demonstrated greater colony counts at 96h h following exposure tothe bacterium.Histopathological examinations demonstrated that lung cells became widened
　　with thickening of the bronchiolar lumen and narrowing of alveolar septa.Exudate inmacrophages resulted in significant increase in lung congestion andsevere consolidation.Themain lesions in the brain were characterized by edema of nerve cells and non-suppurativeencephalitis, indicating that S.pluranimalium is capable of inducing brain damage by crossingthe 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 dogs
　　Microbiological and epidemiological studies were conducted for the examination ofbacterial species, isolated from upper respiratory tract of dogs from Animal Clinics of NanjingAgricultural 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 culturedon culture media such as TSB, LB, blood agar and MacConkeys agar.S.pseudintermediuswas 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％) andStaphylococcus hominis (3.12％).The isolated organisms S.pseudintermedius and S.pluranimalium were subjected to commonly used antibiotics for antibiogram and determinationof resistant drugs.S.pseudintermedius showed antimicrobial resistance to gentamicin(70.83％), tetracycline (50％) and oxacillin (45.83％), while S.pluranimalium hadantimicrobial resistance to enrofloxacin (71.42％), gentamicin (57.14％) and trimethoprim
　　sulfamethoxazole (42.85％).
　　3: Pathogenicity potential ofS.pseudintermedius isolated from dogs with infection signs
　　Experiments were performed to determine the pathogenicity characteristics of S.
　　pseudintermedius isolated from dogs with infection signs in mice.The research was mainlyfocused on clinical symptoms, bacterial loads, histopathological changes and virulence factors.BALB/c mice were intranasaly inoculated with S.pseudintermedius.The results showed thatthe bacteria can cause serious neurologic symptoms including walking in circles and bendingof neck.S.pseudintermedius infection produces obvious histopathological changes in lungsand 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 Ⅱ-Ⅲ, 2 cardedSCCmec Ⅰ and 3 carried SCCmec types Ⅲ 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, China
　　Nasopharyngeal swabs were collected from 6 dogs with severe respiratory syndrome at theAnimal Clinics of Nanjing Agricultural University in 2014.One viral strain was isolated fromSPF embryonated chicken eggs.Sequencing analysis of hemagglutinin (HA) andneuraminidase (NA) genes showed that the CIV isolate shared high similarity (above 98 ％) tothe H3N2 viruses from dogs in China.After the comparison of gene sequence, four uniquemutations 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 theywere in the same evolutionary branching.The result provided a foundation for furtherinvestigation of biological characteristics of CIV.
　　5: Canine influenza virus coinfection with S.pseudintermedius enhances bacterial
　　colonization, virus load and clinical presentation
　　Experiments were conducted to investigate whether synergism exists between canineinfluenza virus and S.pseudintermedius (Sp).For this purpose, BALB/c mice were dividedinto various groups and were inoculated with CIV only, Sp only or both agents (CIV/Sp).Themice in the CIV/Sp co-infection group were challenged with S.pseudintermedius three daysafter CIV infection.During the experiments more severe clinical signs were observed in theCIV/Sp group, and the body weights of the mice in the co-infection group decreasedsignificantly as compared to those of the mice in the CIV-only as well as Sp-only groups.CIVinoculation increased bacterial colonization, whereas secondary infection with S.pseudintermedius elevated the viral RNA titres of CIV in tissues.The histological studiesshowed that lesions in the brain, spleen and lung were more severe in the CIV/Sp group than inthe singly treated groups.Infection in all groups i.e CIV alone, Sp alone or coinfectionstimulated a significantly higher release of cytokines, such as IFN-γ, IL-6, TNF-α andlymphotactin (Lptn), than was observed in the non-infected group.Moreover, the levels ofIFN-γ in the spleen and lung were higher in the CIV/Sp group compared to the CIV-only andSp-only groups.Our findings provide the first demonstration that coinfection with CIV and S.pseudintermedius may synergistically worsen clinical signs and lesion.
　　Key words: canine infectious respiratory disease; Streptococcus pluranimalium;
　　Staphylococcus pseudintermedius; canine influenza virus (CIV); pathogenicity; coinfection。