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Arch Virol 2005 150 1493 1504 DOI 10 1007 s00705 005 0533 x Investigation into the causes of canine infectious respiratory disease antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations K Erles and J Brownlie Department of Pathology and Infectious Diseases The Royal Veterinary College North Mymms U K Received November 4 2004 accepted February 28 2005 Published online April 21 2005 c Springer Verlag 2005 Summary Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease CIRD Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter Additional samples were collected during outbreaks of CIRD The swabs were examined by virus culture and PCR for canine parainfluenza virus canine adeno virus canine herpesvirus CHV and canine respiratory coronavirus CRCoV Furthermore the prevalence of antibodies to CHV and CRCoV was determined During this study CIRD was reported mainly in one of the two kennels investigated In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus which coincided with two outbreaks of respiratory disease CHV antibody responses were detected throughout the year In the other kennel which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels Virus culture failed to detect any viruses in either kennel Introduction Canine infectious respiratory disease CIRD or kennel cough is a disease complex which occurs frequently in densely housed dog populations especially after new dogs have been introduced 1 The problem is well known in rehoming and boarding kennels however it is also an important disease of working dogs because it disrupts their training and can even result in temporary closure of 1494 K Erles and J Brownlie kennels Many factors contribute to the aetiology of CIRD including viruses and bacteria as well as stress due to mixing and housing in an unfamiliar environment Vaccines are available for some of the microorganisms involved in the disease such as canine parainfluenza virus CPIV canine adenovirus type 2 CAV 2 and the bacterium Bordetella bronchiseptica The vaccines have been shown to prevent disease upon experimental challenge with these agents 13 nevertheless CIRD has been reported in many kennels that are using regular vaccination This may suggest that other microorganisms are present in this disease complex Canine herpesvirus CHV has been detected in dogs with CIRD 3 the importance of this infection in the pathogenesis of the disease however has not yet been determined In other species such as cats horses and cattle the involvement of herpesviruses in respiratory disease is well established 11 18 19 CHV has been shown to be able to replicate in the respiratory tract of dogs 2 and has also been detected by PCR in a variety of canine tissue samples 4 In a study of the English pet dog population by Reading et al 15 using an ELISA 88 of dogs were found to be positive for CHV IgG The prevalence of antibodies to CHV in the adult dog population in Belgium was found to be 45 8 17 and a similar prevalence was detected in The Netherlands 39 3 16 CHV infections therefore appear to be common amongst adult dogs Whereas Rijsewijk et al did not find an increase in the number of seropositive dogs after a stay at a boarding kennel an investigation involving dogs at a rehoming centre in London showed that the percentage of positive dogs increased distinctly amongst dogs that had stayed at the kennel for five weeks or longer 7 Experimental infection of dogs with CHV has been shown to cause mild clinical symptoms of rhinitis and pharyngitis 2 or to result in kennel cough 12 Recently a new canine coronavirus was detected in dogs with CIRD housed at a rehoming centre 6 This canine respiratory coronavirus CRCoV was shown to be most similar to bovine coronavirus and human coronavirus OC43 and therefore to be distinct from the canine coronavirus that causes enteritis In that study dogs that were positive for antibodies to CRCoV on arrival in the kennel were less likely to develop CIRD Coronaviruses are known to cause respiratory disease in humans cattle and poultry 8 9 11 however the role of CRCoV in the aetiology of CIRD requires further investigation The study presented here aimed to identify viruses present in dogs at two training centres for working dogs over a period of one year Both kennels had experienced several outbreaks of CIRD during recent years Materials and methods Study population The investigation was carried out at two training centres located in southeast England Kennel A and central England Kennel B All dogs were from the same breeding centre from where they had been placed into families at six to eight weeks of age All dogs were vaccinated against distemper CAV 2 canine parvovirus and Leptospira interrogans At approximately one year of age the dogs then were transferred into the training centres Day one or day of entry CIRD and antibodies to CRCoV and CHV 1495 in this investigation was defined as the day dogs entered the training centre at which time they underwent a routine examination by a veterinary surgeon Dogs at Kennel A received vaccines Nobivac DHPPi and Nobivac Lepto Intervet UK containing distemper CAV 2 canine parvovirus CPIV and Leptospira interrogans Dogs at Kennel B were given a vaccine against distemper CAV 2 canine parvovirus CPIV and Leptospira interrogans Nobivac DHPPi and Nobivac Lepto Intervet UK as well as an additional intranasal vaccine against CPIV and Bordetella bronchiseptica Nobivac KC Intervet UK The target was to follow 25 dogs from each kennel for a period of one year All dogs were therefore taken on to the study on their day of entry Dogs that left the kennel during the study were replaced by new dogs entering the kennel on the following intake date Fur thermore samples were also collected from additional dogs at these kennels during outbreaks of CIRD Sample collection All sampling was approved by the ethics committee responsible for the kennels involved in this study A blood sample was obtained from all dogs on the study on the day of entry and every four weeks thereafter Tonsillar swabs were taken on the day of entry and every three months thereafter From dogs that were not on the study but showed clinical symptoms of CIRD both a blood sample and a swab were taken at the time of disease as well as four weeks after All samples were transported on ice to the laboratory within 24 h Blood samples were centrifuged to obtain serum samples that were stored at 20 C Virus transport swabs Bibby Sterilin Stone UK were used to take swabs from the pharyngeal tonsil of the dogs Cells were detached from the swab and suspended in the transport medium by vigorous mixing The transport buffer containing the cells was then split into three parts and stored at 70 C for subsequent RNA extraction DNA extraction and virus culture In total 340 serum samples and 173 tonsillar swabs were collected from a total of 77 dogs at Kennel A from June 2001 to July 2002 In December 2002 after the study had finished a further outbreak of CIRD occurred at Kennel A and samples were obtained from 13 dogs during the outbreak as well as follow up samples from 11 dogs four weeks later At Kennel B in total 259 serum samples and 119 swabs were collected from a total of 62 dogs from July 2001 to July 2002 Detection of antibodies to CRCoV and CHV The ELISA for CRCoV antibodies was performed using a bovine coronavirus antigen as described previously 6 Briefly the serum samples were routinely tested at a dilution of 1 100 For 28 samples an endpoint titration was performed starting at 1 100 followed by two fold dilutions until a dilution of 1 3200 The immunofluorescence method for the detection of antibodies to CHV using CHV infected MDCK cells has been described elsewhere 7 Serum samples were tested at a dilution of 1 30 DNA and RNA extraction and PCR For DNA extraction using the Qiamp tissue kit Qiagen Crawley UK the transport buffer obtained from tonsillar swabs was centrifuged at 13000 rpm and the resulting cell pellet was resuspended in 200 l of its transport buffer The extraction was performed as recommended by the manufacturer s protocol for cultured cells RNA extraction was performed on swab samples centrifuged as described above The cell pellet was resuspended in 1 ml of TriReagent Sigma Poole UK and the extraction was performed according to the manufacturer s protocol for cultured cells 1496 K Erles and J Brownlie Reverse transcription was performed using ImPromII reverse transcriptase Promega Southampton UK with the provided buffer and 5 l of RNA in a total volume of 25 l For PCR 2 l of cDNA or DNA respectively was used The nested PCR for CRCoV was carried out using the primer pairs Sp1 Sp2 and Sp3 Sp4 directed to the coronavirus spike gene as described previously 6 The primers and methods for the PCR detection of CPIV CAV and CHV were the same as described in 7 Cell culture and virus isolation method Virus isolation was attempted on canine adult lung fibroblasts passages three to seven that were derived from a tissue sample obtained in a previous study 7 and MDCK cells All cells were maintained in MEM Sigma Poole UK containing 100 U ml of penicillin Sigma 0 1 mg ml of streptomycin Sigma and 20 fetal calf serum for canine adult lung fibroblasts and 10 fetal calf serum for MDCK cells Canine adult lung fibroblasts and MDCK cells were inoculated with 500 l of tonsillar swab sample and incubated for 1 h at 37 C The inoculum was then removed and maintenance medium was added to the cells The cultures were passaged weekly for up to three weeks and examined daily for cytopathic effect Statistical analysis Analysis of categorical data was performed using Fisher s exact test For comparison of two means a t test was used P values below 0 05 were considered to be statistically significant Results Prevalence of respiratory disease During the study period from June 2001 to July 2002 most cases of CIRD at Kennel A were reported in October and November 2001 whereas only few dogs were affected during the other months A further outbreak occurred in December 2002 At Kennel B respiratory disease was only present in July and August 2001 and no more cases were reported until the end of the study in July 2002 Figs 1 and 2 At Kennel A 13 out of 35 37 1 female study dogs developed CIRD in contrast to 28 out of 42 66 7 male dogs on the study p 0 0123 At Kennel B three out of 29 10 3 female and four out of 33 12 1 male dogs showed clinical signs of CIRD during the study period p 1 Dogs that developed CIRD had stayed at Kennel A on average for 86 96 days 13 to 275 days The average length of stay of dogs that were present during the same month as the dogs that developed CIRD but remained healthy was 87 21 days 1 to 316 days The difference between the mean stay of dogs with and without CIRD was found to be statistically not significant t test p 0 98 Due to the low number of cases of CIRD at Kennel B the comparison of the mean length of stay was not performed for this kennel CRCoV serology In total twelve out of 54 serum samples 22 2 obtained from dogs on the day they entered Kennel A were positive for antibodies to CRCoV Table 1 shows the CIRD and antibodies to CRCoV and CHV 1497 Fig 1 Antibody response to CRCoV at Kennel A top and Kennel B bottom The black bars represent the number of seroconversions to CRCoV in the respective month the grey bars represent the number of dogs with clinical symptoms of CIRD The line denotes the total percentage of dogs with antibodies to CRCoV in the respective month number of antibody positive dogs entering the kennel each month As shown in Fig 1 seroconversions to CRCoV occurred from November 2001 to February 2002 leading to an increase of the percentage of CRCoV antibody positive dogs to a maximum of 83 percent in December 2001 The subsequent drop in the percentage of positive dogs was due to CRCoV antibody positive dogs leaving the study Samples collected in December 2002 and January 2003 showed that eight dogs seroconverted to CRCoV in January 2003 At Kennel B 32 out of 59 dogs 54 2 were positive for antibodies to CRCoV on the day they entered the kennel Table 1 shows the distribution of antibody positive dogs on the day of arrival from July 2001 to July 2002 Initially the prevalence of CRCoV antibodies in dogs entering Kennel B was very high leading to an overall percentage of positive dogs of 80 to 90 percent Subsequently CRCoV antibody positive dogs were leaving the study and new dogs arriving at the kennel were less frequently positive leading to a continuously declining percentage of 1498 K Erles and J Brownlie Fig 2 Antibody response to CHV at Kennel A top and Kennel B bottom The black bars represent the number of seroconversions to CHV in the respective month the grey bars represent the number of dogs with clinical symptoms of CIRD The line denotes the total percentage of dogs with antibodies to CHV in the respective month positive dogs Fig 1 Only one dog showed a seroconversion to CRCoV during the study period Serum samples taken from the same dogs at different time points were analysed by titration to assess if the titres declined over time Table 2 Paired serum samples from seven dogs at Kennel A consisting of the first CRCoV antibody positive sample at seroconversion and a follow up sample three to five months later were analysed by CRCoV antibody titration The titres at seroconversion ranged from 200 to 1600 Of the seven dogs five remained positive two titres remained stable whereas in three dogs the titres decreased two fold Two dogs with titres of 400 and 200 at seroconversion were negative when tested three months later Titration of seven serum samples from dogs that had been positive for an tibodies to CRCoV on entry into Kennel B revealed titres ranging from 400 to 1600 Follow up samples from six to eight months later showed that the titres had reduced four fold in two dogs in three dogs the titres had decreased two fold and in two dogs they remained unchanged CIRD and antibodies to CRCoV and CHV 1499 Table 1 Number of dogs positive for antibodies to CRCoV or CHV in Kennel A and Kennel B on the day of entry Number of samples in which antibodies were detected total samples tested CRCoV CHV Kennel A Kennel B Kennel A Kennel B Jun 01 0 5 0 0 2 5 0 0 Jul 01 0 2 8 9 0 2 1 9 Aug 01 1 6 2 3 2 6 0 1 Sep 01 1 7 4 5 3 7 0 5 Oct 01 1 8 2 2 1 8 0 2 Nov 01 0 0 7 8 0 0 1 7 Dec 01 0 1 1 3 0 1 0 2 Jan 02 1 4 3 8 0 4 0 5 Feb 02 1 4 0 0 0 4 0 0 Mar 02 3 5 0 0 0 5 0 0 Apr 02 1 6 1 6 0 6 0 6 May 02 3 5 0 3 1 5 0 3 Jun 02 0 1 4 12 0 1 2 12 Jul 02 0 0 0 0 0 0 0 0 Table 2 CRCoV antibody titres of paired serum samples Dog Training Serum Serum Time between number kennel sample 1 sample 2 samples in months 1 Kennel B 400 100 8 2 Kennel B 1600 800 8 3 Kennel B 1600 800 7 4 Kennel B 800 200 7 5 Kennel B 800 400 7 6 Kennel B 400 400 7 7 Kennel B 1600 1600 6 8 Kennel A 800 400 5 9 Kennel A 200 200 5 10 Kennel A 400 200 3 11 Kennel A 1600 800 3 12 Kennel A 800 800 3 13 Kennel A 200 0 3 14 Kennel A 400 0 3 CHV serology Overall the percentage of CHV antibody positive dogs was 16 7 percent 9 out of 54 on the day of entry into Kennel A Dogs joining the kennel from June 2001 to September 2001 were more likely to be positive than those joining 1500 K Erles and J Brownlie during the following months Table 1 Seroconversions to CHV occurred through out the study period with two peaks in November 2001 and May 2002 Fig 2 Due to antibody positive dogs leaving the study in February and March 2002 the percentage of positive dogs declined but increased back to more than 60 percent in May 2002 following a high number of seroconversions At Kennel B four out of 52 dogs 7 7 had antibodies to CHV on their first day at the kennel The overall prevalence of CHV positive dogs over the study period was 7 9 percent and in total only four dogs seroconverted to CHV Fig 2 Antibody responses in dogs with and without CIRD Out of 28 CRCoV antibody negative dogs at Kennel A that developed CIRD 16 57 1 showed a seroconversion to CRCoV within eight weeks after showing clinical signs Of 36 healthy dogs that were present at the same time as the dogs with CIRD 19 52 8 subsequently showed a seroconversion p 0 8 Out of 16 CHV antibody negative dogs with CIRD at Kennel A five 31 3 produced antibodies to CHV within eight weeks of showing clinical signs whereas of 25 healthy dogs present at the same time four 16 showed a seroconversion p 0 28 Antibody prevalences in dogs from the same litter Dogs that were from the same litter but were placed into different kennels for training were paired to compare their antibody status for CRCoV and CHV Analysis of 11 pairs of dogs for antibodies to CRCoV showed that in eight pairs the dog going into Kennel A was negative whereas its littermate going to Kennel B was positive In one pair both dogs were positive and in two pairs both dogs were negative The analysis of antibodies to CHV revealed that in eight pairs both dogs were negative in one pair both dogs were positive in one pair the dog going into Kennel A was positive and in one pair the dog going into Kennel B was positive Antibody responses in male vs female dogs Male dogs showed seroconversions to CRCoV more frequently 56 3 than female dogs 47 6 and were also more likely to become antibody positive for CHV 71 4 compared to female dogs 56 5 but the differences were statistically not significant p 0 58 for CRCoV p 0 38 for CHV PCR analysis Nested RT PCR for CRCoV was performed on 64 samples obtained from dogs housed at Kennel A and three samples were found to be positive One of the positive samples had been taken during an outbreak of CIRD in September 2001 but was from a dog with no clinical symptoms at that time Two further samples CIRD and antibodies to CRCoV and CHV 1501 were derived from dogs with CIRD during the outbreak in November 2001 All samples tested for CPIV n 35 CAV n 7 and CHV n 54 were negative None of the samples obtained from dogs at Kennel B tested by PCR for CRCoV n 28 CPIV n 18 CAV n 9 or CHV n 26 were found positive Virus culture In total 77 throat swab specimens from dogs at Kennel A were used to inoculate canine adult lung fibroblasts or MDCK cells 41 of these were from dogs without clinical signs of respiratory disease and 36 were from dogs suffering from CIRD Furthermore 44 samples from dogs at Kennel B taken from 37 healthy dogs and seven dogs with CIRD were tested for viral growth on both cell lines None of the cultures from either Kennel A or Kennel B yielded any cytopathic effect Discussion In this study outbreaks of CIRD occurred mostly in one of the two study kennels and most cases there were recorded in autumn and winter Similar to the common cold in humans CIRD appears to be seasonal indicating that environmental factors such as low outside temperatures may play a role Although it has not been proven that cooling of the body surface increases susceptibility to respiratory disease it is feasible that cold temperatures may affect the immune defences of mucosal surfaces of the upper respiratory tract permitting facilitated entry of infectious agents 5 Alternatively cold temperatures may be beneficial for a prolonged survival of infectious agents outside a host thereby increasing the likelihood of transmission Male dogs were more frequently affected by CIRD at one of the kennels while there was no difference at the other kennel possibly due to the low number of cases there However in a recent investigation we carried out at a rehoming centre the prevalence