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1、Department of Health and Animal Well-being, Faculty of Veterinary Medicine,University of Bari, ItalySevere Enteric Disease in an Animal Shelter Associated withDual Infections by Canine Adenovirus Type 1 and CanineCoronavirusA. PRATELLI1,4, V. MARTELLA1, G. ELIA1, M. TEMPESTA1, F. GUARDA2,M. T. CAPUC
2、CHIO2, L. E. CARMICHAEL3and C. BUONAVOGLIA1Addresses of authors:1Department of Health and Animal Well-being, Faculty of VeterinaryMedicine, Strada Casamassima km 370010 Valenzano (Bari), Italy;2Department of AnimalPathology, Faculty of Veterinary Medicine, Via Leonardo da Vinci 44 Torino, Italy;3The
3、 JamesA. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Ithaca 14853,NY, USA;4Corresponding author; E-mail: a.pratelliveterinaria.uniba.itWith 2 figures and 1 table(Received for publication October 12, 2000)SummaryAn outbreak of dual infection in dogs with canine a
4、denovirus type 1 (CAV-1) and caninecoronavirus (CCV) infection is reported in an animal shelter that comprised approximately 200adults stray dogs and 30 puppies. Twenty puppies died 78 days after the onset of the clinical signs(severe enteritis, leucopoenia, respiratory distress and dehydration). Bo
5、th CAV-1 and CCV wereisolated from tissue or swab samples. Antibodies to CCV and, at high levels, to CAV-1 also weredetected in several puppies. The principal histological findings were atrophy of small intestinal villi,lymphoid depletion, hepatitis and bronchopneumonia. The persistence of CCV in th
6、e faeces,observed by the polymerase chain reaction assay, was longer than previously reported. Resultsdemonstrated the serious consequences which may occur with dual infections by CAV-1 and CCVin assembled groups of dogs that are housed in poorly managed kennels with inadequate vaccinationprogrammes
7、.IntroductionCanine 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
8、 transient corneal opacity, blue eye, whichmay 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 isobserved more frequently in unvaccinated populations, especially in puppies less than
9、1 year old. Clinical signs of uncomplicated CAV-1 infection usually lasts 57 days, withrapid recovery; however, they may last longer in dogs with concurrent infections, such ascanine distemper virus (CDV2) or, rarely, in animals that develop chronic active hepatitis(Green, 1990; Kobayashi et al., 19
10、933). Although ICH occurs sporadically, the disease hasbeen well controlled since the 1950s when attenuated viral vaccines became available(Appel, 1987a). Serological surveys conducted in various parts of the world prior to theJ. Vet. Med. B 48, 385392 (2001)? 2001 Blackwell Wissenschafts-Verlag, Be
11、rlinISSN 09311793U. S. Copyright Clearance Center Code Statement: 09311793/2001/48050385 $15.00/0www.blackwell.de/synergywidespread use of vaccines indicated that the seroprevalence of CAV-1 antibodies rangedfrom 30 to 60% in the dogs tested (Rubarth, 1947; Brunner et al., 1951; Cabasso, 1953;Sasaki
12、 et al., 1956).Canine coronavirus (CCV) infection is the causative agent of a generally mild, usuallyself-limiting enteric illness in dogs characterized by sudden onset of vomiting andmalodorous diarrhoea. Although CCV infections appear to be mainly asymptomatic,young puppies may be more severely af
13、fected as well as dogs with mixed infections withother viruses or bacteria (Hoskins, 1998).This article describes an outbreak of CAV-1 infection in puppies in an animal shelterin Bari, Italy, with concurrent infection by canine coronavirus (CCV).Materials and MethodsAnimals and clinical findingsThe
14、puppies were located in a kennels which accommodates approximately 200 adult straydogs and 30 puppies less than 4 months old. The sanitary conditions of the kennels were poor; therewas severe overcrowding, with 1520 animals in each room which had area 1213 m2. Although thekennels were washed daily w
15、ith tap water, detergents and disinfectants were only applied irregularly.In addition, vaccination schedules were not systematically carried out and none of the sick puppieshad been vaccinated.In the present outbreak, puppies developed severe enteritis that was sometimes haemorrhagic,shortly after t
16、heir introduction into the kennels. There was no vomiting. The puppies examined(n ? 15) had a moderate leucopoenia, dehydration, abdominal tenderness and were reluctant tomove. Twenty puppies died 78 days after the onset of clinical signs. The puppies that recovered,about 10 days after the onset of
17、the enteritis, had moderate to severe dehydration lasting 13 weeks,and three puppies developed a bilateral keratitis (blue eyes), respiratory distress, cough andaccelerated respiratory rates.Sample collectionsThe collection of the samples for laboratory study could not be carried out systematicallyb
18、ecause 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 atthe onset of enteritis and, thereafter, at 47 day intervals until the puppies had either died orrecovered. Over the 3
19、7 day observation period, 1015 puppies died, but necropsy examinationswere only possible on two animals (no. 19, 31).Histological examinationsTissue samples were obtained from the lungs, small intestine, mesenteric lymph nodes andlivers of the dead puppies (no. 19, 31) and fixed in 10% buffered form
20、alin. Fixed tissue forhistological examination were cut in 5 lm sections, mounted on glass slides, and stained withhaematoxylin and eosin (HE).Virus isolationFaecal and nasal swabs were collected from the sick puppies for a period of 1 month at47 day intervals. Nasal, faecal, and ocular swab samples
21、, as well as tissue samples from the smallintestine, rectum and liver, were collected from the dead puppies. MadinDarby canine kidney(MDCK) and the A-72 dog cell lines were used for virus isolation attempts. Cells were propagated inDulbecco minimal essential medium (D-MEM) that contained 10% (v/v) f
22、oetal bovine serum. Swaband tissue samples were homogenized (10% w/v) in D-MEM and centrifuged at 4000 g for 20 minat 4?C. The supernatant portion of each sample was then treated with antibiotics (5000 IU/ml386PRATELLIet al.penicillin, 2500 lg/ml streptomycin, 10 lg/ml amphotericin) for 30 min at 37
23、?C, and inoculatedonto partially confluent monolayers of A-72 and MDCK cells. The inoculated cell cultures werethen incubated at 37?C in a 5% CO2incubator and observed daily for cytopathic effects. Ifcytopathic effects were absent after 4 days of incubation, cell cultures were frozen and thawed thre
24、etimes and four additional passages were made. Inoculated A-72 cells were also examined at 2-dayintervals by indirect immunofluorescence tests using CCV monoclonal antibodies (generouslysupplied by Dr Gilles Chappuis, Merial, France) and a canine parvovirus (CPV-2) antiserum. Cellcultures which deve
25、loped cytopathic effects were stained with HE to reveal the presence of theinclusion bodies.Nested-polymerase chain reaction for CCVFaecal and nasal samples collected at the onset of illness, and at 47 day intervals thereafterfor 37 days, were examined for CCV in a nested-polymerase chain reaction (
26、n-PCR) assay, aspreviously reported (Pratelli et al., 1999a). Briefly, genomic RNA was extracted from 1 ml of thesupernatant fraction of each sample, using the Rneasy Total RNA Kit (Qiagen GmbH, Hilden,Germany4). The target sequence for amplification was a segment of the gene encoding forthe transme
27、mbrane protein M of CCV. The above sequence of 230 bp straddles nucleotides535 and 746. The following primers were used: CCV1 (5-TCC AGA TAT GTA ATG TTCGC-3), CCV2 (5-TCT GTT GAG TAA TCA CCA GCT-3) and CCV3 (5-GGT GTC ACTCTA ACA TTG CTT-3).CAV endonuclease analysisThe following CAV strains were exam
28、ined: (1) CAV-1, field strain (Buonavoglia et al., 1993);(2) CAV-2, Toronto A26/61 strain (Appel, 1987a5); (3) puppy isolate (no. 30); (4) puppy isolate(no. 19). Endonuclease analysis of CAV DNA was carried out as previously reported (Buonavogliaet al., 1993). Briefly, DNA was extracted from MDCK ce
29、lls with 80% cytopathic effects usingphenol/chloroform and isoamyl alcohol mixture. The digestion was performed with HpaII and PstIendonucleases (Amersham Pharmacia6Biotech, Milan, Italy). A typical reaction, containing 23 ll ofDNA in a 25-ll reaction, and 810 units of enzyme per microgram of DNA, w
30、as applied. Thefragments of the digestion were separated by electrophoresis in a 1.2% agarose gel containing Tris-acetate (90 mMpH 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 UVtransluminat
31、or at 302 nm.Serological studiesSera were not available from the dogs prior to the development of disease; however, serumsamples were collected from 10 dogs at the onset of the clinical signs, and after 12 and 34 days. Allsera were tested for CAV-1, CCV and CDV antibodies by conventional serum neutr
32、alization testsusing MDCK cells for CAV-1, A-72 cells for CCV and VERO cells for CDV. CPV-2 antibodieswere evaluated in haemoagglutination inhibition test.ResultsGross post-mortem examination of the two puppies that were available for necropsyrevealed haemorrhagic enteritis involving the entire smal
33、l intestine. Necrosis andhaemorrhages also were observed in the liver and tonsils of one pup. Gross changeswere not observed in the lungs of either pup. Histologically, there was moderate atrophy ofepithelial villi and an increase in cellularity of the lamina propria in the small intestine.In additi
34、on, lymphoid depletion was observed in the mesenteric lymph nodes. Theprominent microscopic changes intheliver were hydropicdegeneration of thehepatocytes,areas of centrolobular necrosis and the presence of occasional basophilic intranuclear387Dual Infections by Canine Adenovirus Type 1 and Canine C
35、oronavirusinclusion bodies (Fig. 1). The prominent histological lesions in the lungs were moderatepurulent bronchopneumonia with fibrous thickening of the peribronchial tissues.Faecal samples from all puppies examined were negative to CPV-2 by the IFA7test.However, MDCK cells inoculated with nasal,
36、rectal and ocular swabs of one puppy(no. 30) with keratitis, developed a cytopathic effect that consisted of clumps of roundedand refractile cells 4872 h post-inoculation. Similar results were observed in MDCK cellsinoculated with small intestine and rectal swab samples from the other dead puppy(no.
37、 19). Intranuclear inclusion bodies also were observed in MDCK cell monolayers withcytopathic effects. No cytopathic effects or inclusions were observed in MDCK cellsinoculated with liver homogenates from either of the dead puppies. Only A-72 cellsinoculated with rectal swab samples collected at the
38、 onset of the clinical signs from twopuppies (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 swabsamples. CCV shedding was sh
39、own to occur over a period of 737 days in faecal swabs;nasal swabs were positive only for 113 days.Restriction enzyme patterns of the CAV isolates digested with PstI (lines 2, 3, 4and 5) and with HpaII (lines 7, 8, 9 and 10) are shown in Fig. 2. The two isolates (no. 30and 19; lines 4, 5 and 9, 10,
40、respectively) had migration patterns that were similar to thecontrol CAV-1 field strain (lines 2, 7), but were different from CAV-2 (lines 3, 8). The PstIpatterns revealed only a very slight difference between the CAV-1 field strain (line 2) andthe two isolates (no. 30 and 19; lines 4, 5).Serologica
41、l results on 10 of the 15 dogs examined at the initial bleeding, and insurviving dogs at 12 and 34 days after the outbreak was reported, demonstrated highserum neutralization antibody titres to CAV-1 (1:3200). Three puppies had significantincreases (four-fold) in antibody titres during the observati
42、on period. Antibodies toCCV were demonstrated in eight puppies and an increase in antibody titres was observedover the observation period in all puppies tested. Serum neutralization antibody titres toFig. 1. Inclusion bodies typical of adenovirus infection in the liver of puppy no. 19. Haematoxylina
43、nd eosin stain.388PRATELLIet al.CCV were never high (maximum 1:16). There were no elevations in titres to CPV-2, andlow antibodies titres to CDV (1:8) were only observed in two puppies.DiscussionICH is a severe disease of the dog which has rarely been reported during the past fewyears, except for oc
44、casional cases in unvaccinated dogs. On the other hand, CCVinfections are common in dogs, but most are mild, or asymptomatic, except in puppies oradult dogs suffering from stress or other infections (Appel et al., 1979; Appel, 1987b;Hoskins, 1998). The outbreak described in the present report provid
45、es evidence thatCAV-1 is still circulating in the canine population and that serious health problems mayresult where CCV is endemic, management is poor and systematic vaccination is notcarried out. The investigated kennels, an animal shelter, was very poorly managed withrespect to record keeping and
46、 sanitation; moreover, vaccinations had not beenmethodically carried out. The above factors very likely contributed to the severity of theoutbreak of CAV-1 in the kennels. Another unusual finding revealed in this study was thepresence of CCV in the same kennels, often in the puppies that were infect
47、ed with CAV-1.Table 1. Evaluation by PCR of CCV shedding in rectal and nasal swabs from puppiesPuppy no.SamplesCCV shedding (days)Died19arectal12YesnasalND21brectal12Nonasal722rectal24Yesnasal123rectal24Nonasal724rectal7NonasalND25rectal18Yesnasal126rectal18Yesnasal127rectal24Nonasal728rectalnegNona
48、salND29rectal18Yesnasal130a,brectal37Yesnasal131rectal7Yesnasal732rectal13Yesnasal1333rectal13Yesnasal735rectal26Yesnasal7aCAV-1 isolated;bCCV isolated;ND, not done.389Dual Infections by Canine Adenovirus Type 1 and Canine CoronavirusSomeoftheclinicalsignsconstantlyobserved,forexample,theseverehaemo
49、rrhagic enteritis, are not typical of ICH but of other infections such as CPV-2or 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,
50、 8: CAV-2 Toronto/A26/61 strain; lanes 4, 9: puppy no. 30 isolate; lanes 5,10: puppy no. 19 isolate.390PRATELLIet al.CCV infection in dogs is usually self-limited and infected animals usually recoverafter a brief period of illness. Nevertheless, puppies with secondary bacterial infections,parasites
51、or other viral infections may suffer from severe, even fatal, disease (Evermannet al., 1980; Appel, 1987b; Buonavoglia et al., 1993). Dual infections by both CCV andCPV-2 have been reported previously (Appel et al., 1979; Yasoshima et al., 1983; Martinand Zeidner, 1992), and it has been shown that C
52、CV enhances the severity of a sequentialCPV-2 infection. Recently, a severe CCV infection was reported in three puppies thatrecovered from a CPV-2b infection (Pratelli et al., 1999b). Other viruses, such asrotaviruses or caliciviruses, also might enhance the pathogenicity of CAV-1 since mixedinfecti
53、ons 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 anepidemiological point of view and is linked to the duration of the period of virusshedding in the faeces. CCV shedding in faeces has be
54、en reported from 6 to 14 dayspost-infection (Keenan et al., 1976; Tennant et al., 1991). In the present study, CCVwas isolated in cell cultures only from the faecal samples collected at the onset of theclinical signs. Therefore, by means of a highly sensitive test (n-PCR) CCV faecalshedding was dete
55、cted for periods up to 37 days. Although not proved, dual infectionsby both CCV and CAV-1 may have influenced the prolonged shedding of CCV in thefaeces of infected puppies.In summary, it is believed likely that the dual infections (CCV/CAV-1) that wereobserved presented clinical signs similar to th
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