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Department of Health and Animal Well being Faculty of Veterinary Medicine University of Bari Italy Severe Enteric Disease in an Animal Shelter Associated with Dual Infections by Canine Adenovirus Type 1 and Canine Coronavirus A PRATELLI 1 4 V MARTELLA 1 G ELIA 1 M TEMPESTA 1 F GUARDA 2 M T CAPUCCHIO 2 L E CARMICHAEL 3 and C BUONAVOGLIA 1 Addresses of authors 1 Department of Health and Animal Well being Faculty of Veterinary Medicine Strada Casamassima km 3 70010 Valenzano Bari Italy 2 Department of Animal Pathology Faculty of Veterinary Medicine Via Leonardo da Vinci 44 Torino Italy 3 The James A Baker Institute for Animal Health New York State College of Veterinary Medicine Ithaca 14853 NY USA 4 Corresponding author E mail a pratelli veterinaria uniba it With 2 figures and 1 table Received for publication October 12 2000 Summary An outbreak of dual infection in dogs with canine adenovirus type 1 CAV 1 and canine coronavirus CCV infection is reported in an animal shelter that comprised approximately 200 adults stray dogs and 30 puppies Twenty puppies died 7 8 days after the onset of the clinical signs severe enteritis leucopoenia respiratory distress and dehydration Both CAV 1 and CCV were isolated from tissue or swab samples Antibodies to CCV and at high levels to CAV 1 also were detected in several puppies The principal histological findings were atrophy of small intestinal villi lymphoid depletion hepatitis and bronchopneumonia The persistence of CCV in the faeces observed by the polymerase chain reaction assay was longer than previously reported Results demonstrated the serious consequences which may occur with dual infections by CAV 1 and CCV in assembled groups of dogs that are housed in poorly managed kennels with inadequate vaccination programmes Introduction Canine adenovirus type 1 CAV 1 is the causal agent of infectious canine hepatitis ICH The disease is characterized by fever often above 40 C apathy anorexia abdominal pain or tenderness vomiting and diarrhoea Dogs may develop broncho pneumonia conjunctivitis photophobia and a transient corneal opacity blue eye which may occur after clinical recovery as result of anterior uveitis and corneal oedema Appel 1987a Green 1990 ICH is now uncommon in vaccinated populations However it is observed more frequently in unvaccinated populations especially in puppies less than 1 year old Clinical signs of uncomplicated CAV 1 infection usually lasts 5 7 days with rapid recovery however they may last longer in dogs with concurrent infections such as canine distemper virus CDV 2 or rarely in animals that develop chronic active hepatitis Green 1990 Kobayashi et al 1993 3 Although ICH occurs sporadically the disease has been well controlled since the 1950s when attenuated viral vaccines became available Appel 1987a Serological surveys conducted in various parts of the world prior to the J Vet Med B 48 385 392 2001 2001 Blackwell Wissenschafts Verlag Berlin ISSN 0931 1793 U S Copyright Clearance Center Code Statement 0931 1793 2001 4805 0385 15 00 0 www blackwell de synergy widespread use of vaccines indicated that the seroprevalence of CAV 1 antibodies ranged from 30 to 60 in the dogs tested Rubarth 1947 Brunner et al 1951 Cabasso 1953 Sasaki et al 1956 Canine coronavirus CCV infection is the causative agent of a generally mild usually self limiting enteric illness in dogs characterized by sudden onset of vomiting and malodorous diarrhoea Although CCV infections appear to be mainly asymptomatic young puppies may be more severely affected as well as dogs with mixed infections with other viruses or bacteria Hoskins 1998 This article describes an outbreak of CAV 1 infection in puppies in an animal shelter in Bari Italy with concurrent infection by canine coronavirus CCV Materials and Methods Animals and clinical findings The puppies were located in a kennels which accommodates approximately 200 adult stray dogs and 30 puppies less than 4 months old The sanitary conditions of the kennels were poor there was severe overcrowding with 15 20 animals in each room which had area 12 13 m 2 Although the kennels were washed daily with tap water detergents and disinfectants were only applied irregularly In addition vaccination schedules were not systematically carried out and none of the sick puppies had been vaccinated In the present outbreak puppies developed severe enteritis that was sometimes haemorrhagic shortly after their introduction into the kennels There was no vomiting The puppies examined n 15 had a moderate leucopoenia dehydration abdominal tenderness and were reluctant to move Twenty puppies died 7 8 days after the onset of clinical signs The puppies that recovered about 10 days after the onset of the enteritis had moderate to severe dehydration lasting 1 3 weeks and three puppies developed a bilateral keratitis blue eyes respiratory distress cough and accelerated respiratory rates Sample collections The collection of the samples for laboratory study could not be carried out systematically because of the uncontrolled movement of dogs within the kennels and poor record keeping Nevertheless blood samples and nasal and rectal swab specimens were obtained from 15 puppies at the onset of enteritis and thereafter at 4 7 day intervals until the puppies had either died or recovered Over the 37 day observation period 10 15 puppies died but necropsy examinations were only possible on two animals no 19 31 Histological examinations Tissue samples were obtained from the lungs small intestine mesenteric lymph nodes and livers of the dead puppies no 19 31 and fixed in 10 buffered formalin Fixed tissue for histological examination were cut in 5 lm sections mounted on glass slides and stained with haematoxylin and eosin HE Virus isolation Faecal and nasal swabs were collected from the sick puppies for a period of 1 month at 4 7 day intervals Nasal faecal and ocular swab samples as well as tissue samples from the small intestine rectum and liver were collected from the dead puppies Madin Darby canine kidney MDCK and the A 72 dog cell lines were used for virus isolation attempts Cells were propagated in Dulbecco minimal essential medium D MEM that contained 10 v v foetal bovine serum Swab and tissue samples were homogenized 10 w v in D MEM and centrifuged at 4000 g for 20 min at 4 C The supernatant portion of each sample was then treated with antibiotics 5000 IU ml 386 PRATELLI et al penicillin 2500 lg ml streptomycin 10 lg ml amphotericin for 30 min at 37 C and inoculated onto partially confluent monolayers of A 72 and MDCK cells The inoculated cell cultures were then incubated at 37 C in a 5 CO 2 incubator and observed daily for cytopathic effects If cytopathic effects were absent after 4 days of incubation cell cultures were frozen and thawed three times and four additional passages were made Inoculated A 72 cells were also examined at 2 day intervals by indirect immunofluorescence tests using CCV monoclonal antibodies generously supplied by Dr Gilles Chappuis Merial France and a canine parvovirus CPV 2 antiserum Cell cultures which developed cytopathic effects were stained with HE to reveal the presence of the inclusion bodies Nested polymerase chain reaction for CCV Faecal and nasal samples collected at the onset of illness and at 4 7 day intervals thereafter for 37 days were examined for CCV in a nested polymerase chain reaction n PCR assay as previously reported Pratelli et al 1999a Briefly genomic RNA was extracted from 1 ml of the supernatant fraction of each sample using the Rneasy Total RNA Kit Qiagen GmbH Hilden Germany 4 The target sequence for amplification was a segment of the gene encoding for the transmembrane protein M of CCV The above sequence of 230 bp straddles nucleotides 535 and 746 The following primers were used CCV1 5 TCC AGA TAT GTA ATG TTC GC 3 CCV2 5 TCT GTT GAG TAA TCA CCA GCT 3 and CCV3 5 GGT GTC ACT CTA ACA TTG CTT 3 CAV endonuclease analysis The following CAV strains were examined 1 CAV 1 field strain Buonavoglia et al 1993 2 CAV 2 Toronto A26 61 strain Appel 1987a 5 3 puppy isolate no 30 4 puppy isolate no 19 Endonuclease analysis of CAV DNA was carried out as previously reported Buonavoglia et al 1993 Briefly DNA was extracted from MDCK cells with 80 cytopathic effects using phenol chloroform and isoamyl alcohol mixture The digestion was performed with HpaII and PstI endonucleases Amersham Pharmacia 6 Biotech Milan Italy A typical reaction containing 2 3 llof DNA in a 25 ll reaction and 8 10 units of enzyme per microgram of DNA was applied The fragments of the digestion were separated by electrophoresis in a 1 2 agarose gel containing Tris acetate 90 mM pH 8 2 EDTA 2 5 mM and boric acid 90 mM at 70 V and 40 mA for 60 min Visualization of the ethidium bromide stained DNA fragments was performed using a UV transluminator at 302 nm Serological studies Sera were not available from the dogs prior to the development of disease however serum samples were collected from 10 dogs at the onset of the clinical signs and after 12 and 34 days All sera were tested for CAV 1 CCV and CDV antibodies by conventional serum neutralization tests using MDCK cells for CAV 1 A 72 cells for CCV and VERO cells for CDV CPV 2 antibodies were evaluated in haemoagglutination inhibition test Results Gross post mortem examination of the two puppies that were available for necropsy revealed haemorrhagic enteritis involving the entire small intestine Necrosis and haemorrhages also were observed in the liver and tonsils of one pup Gross changes were not observed in the lungs of either pup Histologically there was moderate atrophy of epithelial villi and an increase in cellularity of the lamina propria in the small intestine In addition lymphoid depletion was observed in the mesenteric lymph nodes The prominent microscopic changes in the liver were hydropic degeneration of the hepatocytes areas of centrolobular necrosis and the presence of occasional basophilic intranuclear 387Dual Infections by Canine Adenovirus Type 1 and Canine Coronavirus inclusion bodies Fig 1 The prominent histological lesions in the lungs were moderate purulent bronchopneumonia with fibrous thickening of the peribronchial tissues Faecal samples from all puppies examined were negative to CPV 2 by the IFA 7 test However MDCK cells inoculated with nasal rectal and ocular swabs of one puppy no 30 with keratitis developed a cytopathic effect that consisted of clumps of rounded and refractile cells 48 72 h post inoculation Similar results were observed in MDCK cells inoculated with small intestine and rectal swab samples from the other dead puppy no 19 Intranuclear inclusion bodies also were observed in MDCK cell monolayers with cytopathic effects No cytopathic effects or inclusions were observed in MDCK cells inoculated with liver homogenates from either of the dead puppies Only A 72 cells inoculated with rectal swab samples collected at the onset of the clinical signs from two puppies no 21 30 had cytopathic effects at the third and the fourth serial passages respectively The immunofluorescence test was also positive for CCV Table 1 reports the results of n PCR analysis for CCV on faecal and nasal swab samples CCV shedding was shown to occur over a period of 7 37 days in faecal swabs nasal swabs were positive only for 1 13 days Restriction enzyme patterns of the CAV isolates digested with PstI lines 2 3 4 and 5 and with HpaII lines 7 8 9 and 10 are shown in Fig 2 The two isolates no 30 and 19 lines 4 5 and 9 10 respectively had migration patterns that were similar to the control CAV 1 field strain lines 2 7 but were different from CAV 2 lines 3 8 The PstI patterns revealed only a very slight difference between the CAV 1 field strain line 2 and the two isolates no 30 and 19 lines 4 5 Serological results on 10 of the 15 dogs examined at the initial bleeding and in surviving dogs at 12 and 34 days after the outbreak was reported demonstrated high serum neutralization antibody titres to CAV 1 1 3200 Three puppies had significant increases four fold in antibody titres during the observation period Antibodies to CCV were demonstrated in eight puppies and an increase in antibody titres was observed over the observation period in all puppies tested Serum neutralization antibody titres to Fig 1 Inclusion bodies typical of adenovirus infection in the liver of puppy no 19 Haematoxylin and eosin stain 388 PRATELLI et al CCV were never high maximum 1 16 There were no elevations in titres to CPV 2 and low antibodies titres to CDV 1 8 were only observed in two puppies Discussion ICH is a severe disease of the dog which has rarely been reported during the past few years except for occasional cases in unvaccinated dogs On the other hand CCV infections are common in dogs but most are mild or asymptomatic except in puppies or adult dogs suffering from stress or other infections Appel et al 1979 Appel 1987b Hoskins 1998 The outbreak described in the present report provides evidence that CAV 1 is still circulating in the canine population and that serious health problems may result where CCV is endemic management is poor and systematic vaccination is not carried out The investigated kennels an animal shelter was very poorly managed with respect to record keeping and sanitation moreover vaccinations had not been methodically carried out The above factors very likely contributed to the severity of the outbreak of CAV 1 in the kennels Another unusual finding revealed in this study was the presence of CCV in the same kennels often in the puppies that were infected with CAV 1 Table 1 Evaluation by PCR of CCV shedding in rectal and nasal swabs from puppies Puppy no Samples CCV shedding days Died 19 a rectal 12 Yes nasal ND 21 b rectal 12 No nasal 7 22 rectal 24 Yes nasal 1 23 rectal 24 No nasal 7 24 rectal 7 No nasal ND 25 rectal 18 Yes nasal 1 26 rectal 18 Yes nasal 1 27 rectal 24 No nasal 7 28 rectal neg No nasal ND 29 rectal 18 Yes nasal 1 30 a b rectal 37 Yes nasal 1 31 rectal 7 Yes nasal 7 32 rectal 13 Yes nasal 13 33 rectal 13 Yes nasal 7 35 rectal 26 Yes nasal 7 a CAV 1 isolated b CCV isolated ND not done 389Dual Infections by Canine Adenovirus Type 1 and Canine Coronavirus Some of the clinical signs constantly observed for example the severe haemorrhagic enteritis are not typical of ICH but of other infections such as CPV 2 or CCV Fig 2 Restriction patterns of CAV isolates digested with PstI lines 2 3 4 and 5 and with HpaII lines 7 8 9 and 10 Molecular size markers lanes 1 6 Lambda DNA HindIII Lanes 2 7 CAV 1 strain lanes 3 8 CAV 2 Toronto A26 61 strain lanes 4 9 puppy no 30 isolate lanes 5 10 puppy no 19 isolate 390 PRATELLI et al CCV infection in dogs is usually self limited and infected animals usually recover after a brief period of illness Nevertheless puppies with secondary bacterial infections parasites or other viral infections may suffer from severe even fatal disease Evermann et al 1980 Appel 1987b Buonavoglia et al 1993 Dual infections by both CCV and CPV 2 have been reported previously Appel et al 1979 Yasoshima et al 1983 Martin and Zeidner 1992 and it has been shown that CCV enhances the severity of a sequential CPV 2 infection Recently a severe CCV infection was reported in three puppies that recovered from a CPV 2b infection Pratelli et al 1999b Other viruses such as rotaviruses or caliciviruses also might enhance the pathogenicity of CAV 1 since mixed infections have been observed in dogs with enteritis Hammond and Timoney 1983 Marshall et al 1984 The n PCR for CCV led to another result which seems important from an epidemiological point of view and is linked to the duration of the period of virus shedding in the faeces CCV shedding in faeces has been reported from 6 to 14 days post infection Keenan et al 1976 Tennant et al 1991 In the present study CCV was isolated in cell cultures only from the faecal samples collected at the onset of the clinical signs Therefore by means of a highly sensitive test n PCR CCV faecal shedding was detected for periods up to 37 days Although not proved dual infections by both CCV and CAV 1 may have influenced the prolonged shedding of CCV in the faeces of infected puppies In summary it is believed likely that the dual infections CCV CAV 1 that were observed presented clinical signs similar to those of infectious hepatitis CAV 1 but the enteritis was augmented by the presence of both viruses Infection with CAV 1 may also favour the persistence of CCV in the intestine of infected dogs References Appel M J 1987a Canine adenovirus type 1 infectious canine hepatitis virus In Horzinek M C series ed Virus Infections of Vertebrates Vol I Virus Infections of Carnivores pp 29 43 Elsevier Science Publishers Amsterdam The Netherlands Appel M J 1987b Canine coronavirus In Horzinek M C series ed Virus Infections of Vertebrates Vol I Virus Infections of Carnivores pp 115 122 Elsevier Science Publishers Amsterdam The Netherlands Appel M J B J Cooper H 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recovery of canine c