Patent Abstract:
a vaccine composition for vaccinating dogs comprising any one or more of an agent capable of raising an immune response against streptococcus equi sub species zooepidemicus in a dog , an agent capable of raising an immune response against mycoplasma cynos in a dog , and an agent capable of raising an immune response against a chlamydophila in a dog .

Detailed Description:
the association of streptococcus equi sub species zooepidemicus with canine infectious respiratory disease canine infectious respiratory disease ( cird ) is a multi - factorial infection that affects many kennelled dogs despite the wide use of vaccination . current vaccines aim to protect against viral agents and a single bacterial agent , bordetella bronchiseptica . we examined the role of streptococcal species in cird . the isolation and identification of streptococci in the lower respiratory tract of clinically healthy dogs and those with cird were used to correlate the presence of specific streptococcal species with respiratory disease . we show that the presence of s . equi sub species zooepidemicus ( s . zooepidemicus ) is associated with increasing severity of disease in a population of kennelled dogs with endemic cird . cird is an infection that affects dogs of all ages and commonly occurs when large numbers of dogs are housed together in close confinement . the disease has high morbidity with the dry hacking cough characteristic of laryngitis in the early stages , nasal and / or ocular discharges , and variable anorexia and depression , which can progress to tracheobronchitis , pneumonia and even death in more severe cases . the disease has historically been regarded as a complex infection in which combined or sequential challenge with both viral ( cpiv and cav - 2 ) and bacterial agents produces a synergistic enhancement of the clinical scores ( appel and binn , 1987 ). the most common bacterial agent detected during the disease is b . bronchiseptica ( mccandlish et al . 1978 ), but other bacterial species such as pasteurella sp ., mycoplasma sp . and β - haemolytic streptococci ( βhs ) have all been associated with disease ( mccandlish et al ., 1978 ; rosendal , 1978 ; thrusfield et al ., 1991 ). many studies involving bacterial isolation from the upper ( oral and nasal cavity ) and lower respiratory tract ( trachea and lungs ) of both diseased and healthy dogs mention the presence of βhs ( smith , 1967 ; mccandlish et al ., 1978 ; mckieman et al ., 1982 ; azetaka and konishi , 1988 ). however , despite the variety of species of βhs found in the upper respiratory tract of dogs , only a few investigations have focused upon the species of βhs involved in lower airway disease ( garnett et al ., 1982 ; angus et al ., 1997 ). although species of βhs in the canine respiratory tract were noted by biberstein et al ., ( 1980 ) this study neglected to distinguish between carriage in the upper and lower respiratory tract . furthermore , even though isolation was from veterinary hospital patients the reason for referral and therefore any link to specific clinical conditions was omitted . the most common βhs in dogs , s . canis , a lancefield group g streptococcus , is a normal commensal of the genital and respiratory mucosa as well as skin ( timoney , 1987 ; quinn et al ., 1999 ). streptococcus canis ( s . canis ) has previously been isolated from the tonsils of 60 to 73 % of healthy dogs ( smith , 1967 ; sadatsune and moreno , 1975 ; biberstein and hirsh , 1999 ). s . canis causes a variety of sporadic and opportunistic infections in dogs , including pneumonia , septicemia , abscesses , otitis , mastitis , pyometra , proctitis , toxic shock syndrome and necrotising fasciitis ( biberstein and hirsh , 1999 ; quinn et al ., 1999 ). in addition to s . canis βhs of other lancefield groups , such as a , c and e , have also been isolated from dogs ( biberstein et al ., 1980 ). s . zooepidemicus lancefield group c , is found as a commensal of the upper respiratory tract mucosa of mammals ( timoney et al ., 1988 ; quinn et al ., 1999 ). it is associated with several disease syndromes including lower airway disease , foal pneumonia and cervicitis in horses ( chanter , 1997 ; biberstein and hirsh , 1999 ), pneumonia in llamas ( biberstein and hirsh , 1999 ), septicemia and arthritis in pigs ( timoney , 1987 ), mastitis in cows and goats ( timoney et al ., 1988 ), septicemia in poultry , pericarditis and pneumonia in lambs ( timoney , 1987 ), lymphadenitis in guinea pigs ( quinn et al ., 1999 ) and glomerulonephritis in humans ( balter et al ., 2000 ). in dogs s . zooepidemicus has been associated with wound infections , septicemia ( quinn et al ., 1999 ) and acute necrotizing hemorrhagic pneumonia ( garnett et al ., 1982 ). in this study we sought to establish which species of βhs are present in the respiratory tract of both healthy dogs and those with cird . the main study population ( n = 209 , bronchial alveolar lavage , bal ) comprised animals from a well - established re - homimg kennel (˜ 600 dogs ) with a history of endemic cird . on entry to the kennel all dogs were vaccinated with kavak da 2 pip69 ( fort dodge ) a live attenuated vaccine for distemper virus , cav - 2 , cpiv and canine parvovirus and kavak l against leptospirosis . the presence of both canine coronavirus ( crcv ) and b . bronchiseptica has been demonstrated in dogs with cird in this center ( chalker et al ., 2003 ; erles et al ., 2003 ). each week this kennel must sacrifice some dogs for welfare reasons and from these dogs 2 - 3 were selected arbitrarily for sampling . bal samples were taken by the following method from a total of 209 individual dogs over a 2 year period from 1999 to 2001 . within 2 hours of euthanasia the trachea was clamped just above the bifurcation to prevent any tracheal contamination of the lung during sampling . using sterile catheter tubing 50 ml hanks balanced salt solution was then placed into the left apical lung lobe . this lung lobe was then massaged manually for 30 seconds and the bal withdrawn . at euthanasia dogs were also graded for the severity of clinical respiratory score into the following categories : ( 1 ) no respiratory signs , n = 71 ( 2 ) mild cough , n = 37 ( 3 ) cough and nasal discharge , n = 76 ( 4 ) cough and nasal discharge with depression and / or inappetence n = 9 ( 5 ) suppurative bronchopneumonia , n = 16 . after bal sampling a section of lung tissue from the right distal lobe was taken for histological analysis . formalin fixed ( 10 % formalin saline ) tissue blocks were embedded in paraffin , and standard hemtoxylin and eosin stained sections were viewed under a light microscope ( x40 , xioo , x400 ). the presence or absence of intra - alveolar neutrophils was noted . the total number of days each dog spent in the kennel was recorded and time in the kennel was then calculated in weeks . the age and clinical condition on entry into the kennel of each animal was noted and a clinical condition composite score based on nutritional status , coat , demeanor , appetite and a general clinical examination ( temperature , pulse rate , respiration rate ) was graded as follows : good ( 1 ), poor ( 2 ), very poor ( 3 ). an additional dog population was included as a control group that comprised of household pet dogs with clinical respiratory symptoms referred to diagnostic bacteriology at the rvc over a 2 year period ( 1998 to 2000 ) ( n = 71 , bal ). samples from the control group were collected using an endoscopically guided technique as described by cocoran ( 1998 ). all samples in the study were kept at 4 ° c . until bacteriological testing , and testing was performed within 24 h of sampling excepting the calculation of cfu per ml that was performed on frozen bal . a 50 μl volume of bal was plated in duplicate onto columbia blood agar ( oxoid ltd ., hampshire , uk ) plates with 5 % sterile sheep blood , and incubated both aerobically and anaerobically for 24 hrs at 37 ° c . β - haemolytic colonies were identified and then purified to single colonies . gram - positive catalase - negative bacteria were identified as streptococci by colonial and cellular morphology , and then serogrouped by latex bead slide agglutination ( oxoid ltd ., hampshire , uk ) into lancefield groups . isolates were then identified to the species level by biochemical utilization and enzymatic action using the api20strep manual identification kit ( biomerieux uk ltd ., basingstoke , uk ). in order to detect mixed infections 3 colonies from the first 12 dogs in the study were tested by both latex bead slide agglutination and api20strep . serial dilutions of bal in phosphate buffered saline ( sigma - aldrich co . ltd ., dorset , uk ) were plated in triplicate , incubated as described above and the cfu per ml bal calculated . growth of βhs was then graded as follows : none ( o ), & lt ; 100 cfu per ml ( 1 ), 100 to 1000 cfu per ml ( 2 ), and & gt ; 1 000 cfu per ml ( 3 ). a significance level or probability of a type i error ( α ) of 0 . 05 was assumed for all analyses . the presence of s . zooepidemicus with the age , clinical condition on entry to the kennel , weeks in the kennel , the presence of intra - alveolar neutrophils and clinical respiratory scores was analyzed using prism ( version 3 . 0 , graphpad software inc , san diego , usa ) statistical analysis software x 2 testing . the correlation of bacterial growth and respiratory score was determined by use of the combined mean scores for s . zooepidemicus growth for each respiratory score , analyzed with prism one way anova ( non - parametric ) testing . the presence of s . canis , s . zooepidemicus and respiratory disease in the sampled kennelled dogs with time in weeks was also calculated . β - haemolytic streptococci were isolated from both study populations , and isolation from the bal of household pets was markedly different from the 2 kennelled dogs ( 1 . 4 % household , 23 . 9 % kennel , χ 2 analysis *** p = 0 . 000 ). all hs isolates were found to be s . canis or s . zooepidemicus . mixed infections with differing lancefield groups or species were not found , furthermore all individual plates yielded colonies of uniform morphology . both s . canis and s . zooepidemicus were isolated from the kennelled dogs , whereas only a single isolate of s . zooepidemicus and no s . canis were isolated from the household pets . s . zooepidemicus was found to be the predominant hs species in the kennelled dogs ( 92 . 0 %). the carriage of both s . canis and s . zooepidemicus was examined in the kennelled dogs within each grade of clinical respiratory score ( fig1 ). s . canis was present in dogs both with and without clinical scores , and isolation did not increase with disease severity . by contrast , healthy dogs were less likely to have s . zooepidemicus in the lower respiratory tract than diseased animals ( χ 2 analysis , ** p = 0 . 004 ) and the isolation of s . zooepidemicus increased dramatically with increasing clinical respiratory score , from 9 . 7 % in dogs with no symptoms to 87 . 5 % in those dogs with suppurative 2 bronchopneumonia ( χ 2 analysis , *** p = 0 . 000 ). dogs with higher respiratory scores were also more likely to have a greater mean s . zooepidemicus bacterial growth score than clinically healthy dogs ( one way anova analysis *** p = 0 . 000 . r squared = 0 . 194 , f = 22 . 265 ). the age and clinical condition of the animal on entry to the kennel had no affect on the isolation of s . zooepidemicus ( χ 2 analysis , age p = 0 . 341 , clinical condition on entry p = 0 . 295 ). the percentage of dogs with cird in the kennel increased dramatically from 21 . 1 % in week 1 to 70 . 1 % in week 2 , and cird did not decrease in the population until after the fourth week ( fig2 ). although no significant difference was detected , the number of dogs with s . zooepidemicus in the lung increased by 20 . 6 % with time in the kennel from 16 . 7 % in week 1 to 34 . 4 % in week 3 ( fig2 ), whereas no such trend was seen with s . canis . histological analysis revealed that dogs with s . zooepidemicus were more likely to have intra - alveolar neutrophils than those without s . zooepidemicus 2 ( χ 2 analysis , ** p = 0 . 006 ). in dogs with higher bacterial scores , acute suppurative or necrotizing pneumonia with moderate to marked macrophage aggregation was often noted , similar to the findings of garnett et al ., ( 1982 ) in dogs with s . zooepidemicus induced hemorrhagic streptococcal pneumonia ( hsp ). no bacterial cells were apparent on hand e stained sections . in this study we focused upon the species of βhs present in the lower respiratory tract of household and kennelled dogs , with and without respiratory disease . although s . canis is the predominant βhs of the respiratory tract in dogs ( biberstein et al ., 1980 ) and was isolated from the lower respiratory tract of some kennelled dogs in this study , it was not associated with cird in the kennelled dogs . in contrast , an increased isolation of s . zooepidemicus was associated with increasing cird severity . dogs with any respiratory symptoms were more likely to have s . zooepidemicus in the lower respiratory tract than more healthy animals in the kennel and s . zooepidemicus was found in a lower proportion of the household pets than the kennelled dogs . streptococcus equi sub species zooepidemicus has previously been associated with hsp in dogs ( garnett et al ., 1982 ). the hsp syndrome was a severe infection in a closed colony of beagles , in which sudden death ensued without prior clinical scores . necropsy findings included abundant hemorrhagic exudates within the trachea and bronchial tree , with diffuse dark reddening of the lungs . in addition , there were ecchymotic hemorrhages of a range of other tissues . the disease was reproduced by intra - tracheal inoculation with s . zooepidemicus in one dog . interestingly in this study , dogs with higher s . zooepidemicus growth scores were more likely to have intra - alveolar neutrophils and share histological features of the lungs described by garnett et al ., ( 1982 ) in hsp than those dogs with low growth scores . cird has historically been considered a complex disease , involving both bacterial and viral agents . indeed , several other agents have been described in this kennelled population of dogs , including crcv ( erles et al ., 2003 ) and b . bronchiseptica ( chalker et al ., 2003 ). although the pathogenic potential of crcv has not yet been clarified , data by erles et al ., ( 2003 ) shows that crcv predominates in those dogs with mild respiratory disease ( score 2 ) and similarly chalker et al ., ( 2003 ) found that dogs with b . bronchiseptica predominates in those dogs with moderate disease ( score 3 ). we found that streptococcus zooepidemicus is associated more commonly with only the more severe cases of cird ( score 4 - 5 ) indicating it may act as a secondary invader . indeed , βhs species have previously been described as secondary invaders in the cird ‘ complex ’ ( mccandlish et al ., 1978 ). however , it is still not known if s . zooepidemicus plays a primary role in respiratory disease in these animals or merely invades the respiratory tract following damage by other pathogens . epidemiological evidence suggests that in the horse s . zooepidemicus may be a primary pathogen in respiratory disease ( wood et al ., 1993 ; chanter , 1997 ) but it is generally considered to be an opportunistic pathogen ( walker and timoney 1998 ; anzai et al ., 2000 ). even if s . zooepidemicus is not a primary cause of cird in these dogs , the high isolation rate from dogs with suppurative bronchopneumonia ( 87 . 5 %) supports the hypothesis that s . zooepidemicus is responsible for the more severe clinical signs seen in this kennel . the low isolation from household pets ( 1 . 4 %) with respiratory disease indicates this agent may not be a common respiratory infection and could be a problem particular to this kennel . although any previous kennelling was not taken into consideration it is likely that some of the household pet dogs in this study have been kennelled at one time . the role played by s . zooepidemicus in other cases of cird in kennelled dogs has not been ascertained . the isolation of s . zooepidemicus from these dogs increases with time in the kennel , indicating the lungs of these dogs are becoming infected with this bacterium . such infection could be occurring from either sub - clinical infections of the upper respiratory tract or from a single pathogenic strain . a pcr typing system for the gene of the variable m - like szp protein enables the separation of the 15 known sero - types of s . zooepidemicus into five distinct groups , hv1 - 5 ( walker and timoney , 1998 ). analyses with this typing system by anzai et al ., ( 2000 ) found that single clonal variants of s . zooepidemicus are found in the pneumonic equine lung whereas several types are found in the tonsils of healthy horses . it would be of interest to sub - type the s . zooepidemicus isolates involved in this outbreak of cird to determine whether a single clonal variant is present in the diseased population , and also to examine the relationship , if any , that canine s . zooepidemicus isolates have to those causing respiratory disease in horses and other animals . s . zooepidemicus associated pneumonia occurs in horses of all ages and acute hemorrhagic pneumonia in older horses that have been stressed by transportation ( anzai et al ., 2000 ). in this outbreak of cird younger dogs and those in poor clinical condition on entry to the kennel were equally susceptible to infection with s . zooepidemicus as the older dogs and those that were healthy on entry . in this kennel antibiotic therapy is given for a range of infections , and treatment is not routinely given to dogs with cird except in cases of severe bronchopneumonia . it is possible that treatment could have influenced the bacterial spectrum noted in this study . however the examination of natural outbreaks of respiratory disease can provide valuable information that cannot be obtained by other means . cird is known to be a multi - factorial disease involving several agents including cav - 2 , cpiv , b . bronchiceptica and mycoplasma spp . in this kennel in which large numbers of dogs from a variety of locations are brought together and housed , several pathogens are present and the severity of the disease may reflect this . the association of mycoplasma cynos with canine infectious respiratory disease the presence of m . cynos was investigated by culture of the organism and identification by pcr analysis . in a survey of 184 kennelled dogs we have found that the percentage of dogs with m . cynos in the trachea or lung increases with signs of respiratory disease from 10 % in healthy dogs to 31 % in diseased dogs ( fig3 ). we have also noted that respiratory disease increases with time in the kennel and during the first week in the kennel dogs have no detectable m . cynos in the trachea , whereas by the second week 24 % of the 184 dogs were positive for m . cynos in the trachea — indicating 24 % of the population are being infected with this bacterium . a smaller but similar increase was also seen for colonization of the lung ( from 15 % to 23 %) ( see fig4 ). we surveyed 210 dogs by pcr analysis for the presence of chlamydophila . a 218 bp fragment of the 23s rrna gene was amplified from the chlamydophila by the following pcr . reaction conditions , 95 ° c . 5 min (× 1 cycle ), 95 ° c . 30 seconds , 50 ° c . 30 seconds , 72 ° c . 1 minute (× 40 cycles ) and 72 ° c . 5 mins . the pcr reaction mix of 50 μl total , included 5 . 0 μl 10 × magnesium free buffer ( promega ), 1 . 5 mm mgcl 2 ( promega ), 0 . 5 μl ( 0 . 5 units ) taq dna polymerase ( promega ), 0 . 2 mm pcr nucleotide mix ( promega ), 0 . 025 μg forward primer c1 ( 5 ′- gatgccttggcattgataggcgatgaag ga - 3 ′, seq id no : 9 ) and reverse primer c2 ( 5 ′- tggctcatcatgcaaaaggca - 3 ′, seq id no : 10 ), 40 μl water and 2 μl sample tissue dna . a pcr product obtained from 8 dogs was confirmed as a chlamydophila by sequence analysis and comparison of the pcr product to all available sequences in genbank by fasta analysis . the partial sequence of the 23s rrna gene of one such sequence ( dhbci0 ) is shown in fig5 ( seq id no : 1 ). this 218 bp sequence is 99 . 08 % identical to the same region in chlamydophila abortus and 98 . 6 % identical to chlamydophila psittaci and 96 . 3 % identical to chlamydophila felis and on preliminary phylogenetic analysis ( clustal method with megalign ) most sequences cluster in a distinct clade ( fig7 ). the 23s rrna partial sequences of seven other chlamydophila isolates are shown in fig8 ( seq id nos : 2 - 8 ). in this survey we found an increase in the detection of chlamydophila with increasing respiratory disease severity in both the trachea and lung . a slight increase of detection of 10 % was found in tracheal samples ( from 25 % to 34 %). a more dramatic difference was found in detection of chlamydophila in the lung , with an increase from 0 % healthy dogs to 37 . 5 % in dogs with cird ( fig6 ). furthermore , an increase in the total number of dogs that tested positive by pcr for chlamydophila from 25 % in healthy dogs to 50 % in dogs with severe disease was noted ( fig6 ). we found an increased prevalence of canine herpesvirus in dogs with more severe respiratory symptoms ( fig9 ). when monitoring antibody responses to chv over a yearlong period , dogs in a kennel with frequent outbreaks of respiratory disease showed seroconversions to crv more frequently ( 58 . 3 %) than dogs from a comparable kennel with no outbreaks ( 8 . 3 %). angus , j c ., jang , s . s ., hirsh . d . c ., ( 1997 ). microbiological study of transtracheal aspirates from dogs with suspected lower respiratory tract disease : 264 cases ( 1989 - 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