None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
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The authors greatly appreciate funding support of National Applied Research Laboratories, the valuable histological technical support from Miss Shiow-Ling Liao and Yi-Ying Chiu, helpful comments of Dr. Richard J. Montali and Dr. Sarah L.
Poynton, Johns Hopkins University School of Medicine, Baltimore, USA.
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Table 1. RT-PCR primer sequence of the phosphoprotein (P) gene of CDV Onderstepoort, strain(AF378705)
Primer Nucleotide sequence (5’-3’) Nucleotide position
Target Set 1
F2 TAAGGGAATCGAAGATGC 2160-2177 -
R1 CCATCAGCATGCTCACATC 2359-2341 200
R2 GATCCCCCAGTTGACTTG 2585-2568 426
Set 2
P2 (F) ATGTTTATGATCACAGCGGT 2132-2151 429
P2b (F) ATTAAAAAGGG(G/C)ACAGGAGAGAGATCAGCC 2482-2511 78
P1(R) ATTGGGTTGCACCACTTGTC 2560-2540 -
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group of CDV infected dogs. Data is presented as number of positive cases, and percentage of positive cases.
Diagnostic Lesions Shelter (N=20) Clinic (N=32) Chi-square test Intranuclear inclusion
body 11(55%) 28(87%) **
Demyelination, Cerebrum 7(35%) 25(78%) **
Demyelination,
Cerebellum 4(20%) 23(71%) **
Demyelination, Brain stem 6(30%) 25(78%) **
Lymphoid depletion 10(50%) 31(96%) **
Catarrhal enteritis 6(30%) 11(34%) ns
Interstitial pneumonia 11(55%) 26(81%) *
Hyperkeratosis 4(20%) 9(28%) ns
Syncytium 6(30%) 14(43%) ns
**, statistically significant, P<.01; * , statistically significant, P<.05; ns, not statistically significant
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111
112
C 98-5102 NA + - - + - - - NA
C dog1 NA - - + + + + - NA
C 09-997 NA - - + + + + - -
C 09-887 NA - + + + - + + NA
No.
detected
17 21 20 18 15 13 14 14 7
% positive 68 65 62 56 46 40 43 43 25
C:clinic ; CV: clinic treatment with CDV vaccination history; +: positive; -: negative; NA: not available; r
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Table 5. Comparison of the frequency of intranuclear and intracytoplasmic inclusion bodies (INIB/ICIB) in 9 sites in shelter and clinic CDV-infected dogs.
Data presented are number of INIB/ICIB positive dogs, total number of dogs examined, and the percentage of dogs INIB/ICIB positive.
Sites of INIB/ICIB Shelter group Clinic group chi-square test
nos % nos %
Urinary bladder 5/19 26 17/25 68 **
Lymphoid tissues 7/20 35 21/32 65 *
Lung 3/20 15 20/32 62 **
Cerebellum 7/20 35 18/32 56 ns
Cerebrum 4/20 20 15/32 46 ns
Brain stem 6/20 30 13/32 40 ns
Ependymal cells 5/20 25 14/32 43 ns
Alimentary tract 2/20 10 14/32 43 *
Skin 2/20 10 7/28 25 ns
+ positive; - negative; statistically significant * P < 0.05, ** P < 0.01; ns, not significant; nos number of samples
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clinical group of CDV infected dogs (Percentage of different lesions and co-infections /total animal counted
Co-infections /Organ
Co-infections and associated lesions Shelter group, (N=20) (%)
Clinic group (N=32) (%) Co-infections
Virus Adenovirus infection, liver, type I
Adenovirus infection, lung, type II
Potozoa Coccidiosis
Toxoplasmosis
Babesiosis
Mycoplasma Hemobartonellosis
Fungi Aspergillosis 0 3
Suppurative bronchopneumonia
Lung abscess
Heart Myocarditis
Liver Hepatitis
Adrenal gland Nodular hyperplasia of adrenal gland
Hemorrhage and necrosis of adrenal gland
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co-infections and associated lesions, there were no significant differences in frequency of occurrence between shelter and clinic dogs (X2 chi-square test); the exception was interstitial nephritis, which was significantly more common in the shelter dogs than in the clinic dogs.
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Table7. Diagnostic Criteria of CDV cases included in this study, N=52
Single test RT-PCR IHC Demyelination INIB/ICIB
positive 38(73%) 33(63%)
34(65%) 39(75%)
negative 0 1
not available 14(27%); S 2; C12 18(35%); S 13;C5
Combined tests 20
positive 19 negative 1
C: clinic; INIB/ICIB: intranuclear or intracytoplasmic inclusion bodies; IHC: immunohistochemistry;
RT-PCR: Reverse Transcription Polymerase Chain Reaction, S: shelter.
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Figure 1. Comparison between H & E and IHC labelling in two CDV infected dogs.
A, B. Case 58, brain. A. H & E, showing multifocal spongy form of the mid-brain thalamus neuropil B, non-biotin HRP (AEC) with haematoxylin counterstain, showing diffuse immunolabelling of thalamus neuropil. C, D. Case 57, lung. A. H & E, showing interstitial pneumonia with exfoliated pulmonary macrophages and necrotic debris in the alveolar lumen. D, non-biotin HRP (AEC) with haematoxylin counterstain showing intracytoplasmic immunoreactivity in the bronchiolar epithelium and macrophages. All scale bars = 40 μm.
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Figure 2. Eosinophilic intranuclear and intracytoplasmic inclusion bodies in four dogs with CDV. H & E. A, multifocal spongy form of the mid-brain thalamus neuropil with eosinophilic intranuclear inclusion bodies in the sub-ependymal astrocytes (arrowhead), case 58. B, splenic lymphoid depletion and necrosis with eosinophilic intranuclear inclusion bodies (arrowhead) in the white pulp, case 49. C, eosinophilic intranuclear and intracytoplasmic inclusion bodies (arrowhead) in the bladder ballooning mucosa epithelium, case 51. D, eosinophilic intranuclear inclusion bodies (arrowhead) in the gastric mucosa gland, case 50. All scale bars = 10 μm.
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Figure 3. Co-infections and associated lesion in four dogs with CDV. H & E. A, purulent bronchointerstitial pneumonia with smudge 8-10μm adenovirus, basophilic intranuclear inclusion bodies in the pulmonary bronchiolar epithelium (arrow), case 58. B, 10-40μm (body length) microfilaria of Dirofilaria immitis (arrows)deposited in the pulmonary alveolar septa, and capillaries with nodular aggregates of pyogranulomatous epithelioid macrophages, eosinophilic intranuclear inclusion bodies in the macrophages(arrowhead), case 49. C, hepatic cell necrosis with 5-8μm basophilic adenovirus, intranuclear inclusion bodies (arrows) in the hepatocytes, case 51. D, hemorrhagic to necrotizing pneumonia with numerous, 4-8 x 2-4 um, intra-lesional, curvilinear tachyzoites (arrow) free within areas of pulmonary necrotic parenchyma, case 50. All scale bars =10 μm.
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