Biomedical subjects
Publications and source records attributed to Nicola Decaro.
The ability of a modified live canine parvovirus type 2b vaccine to elicit active immunization in pups with maternally derived antibodies (MDA) by intranasal administration was evaluated. The vaccine induced seroconversion in 100% of pups with MDA titers of < or = 80 and in 51.6% of pups with titers between 160 and 320.
An outbreak of canine parvovirus type 2 infection caused by the Glu-426 mutant in 2 litters of pups is reported. The infected pups (n = 6) were monitored daily for evidence of clinical signs and hematological changes and for the evaluation of viral shedding in the feces. The disease induced by the Glu-426 mutant was mild in all the infected pups. Vomiting and hemorrhagic diarrhea were not observed; however, the pups developed mucoid diarrhea (3.5 median days), depression (1.5 median days), and relative leukopenia and lymphopenia (2.5 median days). Fever and loss of appetite were observed only in 2 pups. Virus was detected in the feces for 4.5, 6.5, and 46 median days by hemagglutination, virus isolation on cell cultures, and real-time polymerase chain reaction (PCR), respectively. By real-time PCR, the highest viral DNA titers were detected in the feces of both litters at day 10, reaching median values of more than 10(10) DNA copies/mg of feces.
A case of feline infectious peritonitis (FIP) in an 11-month-old European shorthair cat is reported. The infected cat displayed loss of weight, respiratory distress, ascitis, anemia and died within 15 days after the first appearance of clinical signs. Lesions typical of a mixed form (effusive and non-effusive) of FIP were observed and by RT-PCR a feline coronavirus (FCoV) type I strain was detected in several tissues. The RT-PCR results were confirmed by sequence analysis of the amplified products. Phylogeny carried out on fragments of the M and S genes showed that the FCoV strain segregates with typical type I FCoVs.
We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2 (CPV-2) DNA in the feces of dogs with diarrhea. An exogenous internal control was added to control the assay performance from extraction to amplification. The method was demonstrated to be highly specific and sensitive, allowing a precise CPV-2 DNA quantitation over a range of eight orders of magnitude (from 10(2) to 10(9) copies of standard DNA). The reproducibility of the CPV-2 real-time PCR assay was assessed by calculating the coefficients of variation (CV) intra-assay and inter-assay for samples containing amounts of CPV-2 DNA spanning the whole range of the real-time PCR standard curve. Then, fecal specimens from diarrheic dogs were analyzed by hemagglutination (HA), conventional PCR and real-time amplification. Comparison between these different techniques revealed that real-time PCR is more sensitive than HA and conventional gel-based PCR, allowing to detect low viral titers of CPV-2 in infected dogs.
A novel nested PCR (nPCR) assay is reported on the diagnosis of the feline calicivirus (FCV) infection. The test was performed on 47 ocular and 40 pharyngeal swabs collected from 47 cats with respiratory syndrome; among the 87 samples examined, 18 ocular and 23 pharyngeal swabs were positive in nPCR. The nPCR sensitivity was compared to other diagnostic techniques such as virus isolation on cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR). The nPCR was more sensitive than the virus isolation and RT-PCR; therefore, it can be used for calicivirosis diagnosis in cats.
Two fragments, S66 and S55, of the S glycoprotein of the newly identified canine coronavirus type I (CCoV type I), were expressed in a procariotic system. The purified recombinant proteins of 350 and 366 amino acids in length, respectively, were employed to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of CCoV type I antibodies in dog sera. Four canine sera-positive for CCoV type II, four sera-positive for CCoV type I and 10 negative control sera were examined. Only the sera-positive for CCoV type I strongly reacted with both the proteins, whereas the sera-positive for CCoV type II showed low reactivity in the ELISA test. As CCoV type I seems to be not cultivable in cell cultures, the recombinant fragments of the S protein represent a unique method to study, preliminarily, the immunological and the pathogenetic characteristics of this new virus.
A TaqMan fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies of CCoV standard RNA, and was linear from 10 to 10(8) copies, allowing quantitation of samples with a wide range of CCoV RNA loads. A total of 78 faecal samples of diarrhoeic dogs were tested simultaneously by conventional and fluorogenic RT-PCR: 29 were negative by both techniques, whereas 27 tested positive by conventional RT-PCR and 48 by the established CCoV fluorogenic assay. One sample, which was positive by conventional RT-PCR, gave no signal in the fluorogenic assay. In addition, by the fluorogenic assay CCoV shedding in the faecal samples of an experimentally infected dog was monitored for 28 days. The high sensitivity, simplicity and reproducibility of the CCoV fluorogenic RT-PCR assay, combined with its wide dynamic range and high throughput, make this method especially suitable for efficacy trials on CCoV vaccines.
Fecal secretory immunoglobulin A (IgA) antibodies in dogs infected or vaccinated with canine coronavirus (CCV) were evaluated by an enzyme-linked immunosorbent assay. The study was carried out with 32 fecal samples collected just before inoculation and at 28 days postinoculation. Five groups were studied: naturally infected dogs, experimentally infected dogs, dogs inoculated with a modified live (ML) CCV vaccine by the intramuscular route, dogs inoculated with an ML CCV vaccine by the oronasal route, and dogs given an inactivated CCV vaccine. Both the naturally and the experimentally infected dogs developed high levels of fecal IgAs. Interestingly, dogs inoculated with the ML CCV vaccine by the oronasal route developed levels of fecal IgA that were higher than those observed in the dogs inoculated with the same CCV vaccine by the intramuscular route or those observed in dogs inoculated with the inactivated vaccine. A relationship between the level of fecal IgAs to CCV and the degree of protection against CCV infection was observed.
Sixty-nine fecal samples from diarrheic puppies were examined by reverse transcription-PCR assays for the M and the S genes of canine coronaviruses (CCoVs). The isolates in 10 samples were recognized as CCoV type I, and the isolates in 6 samples were recognized as CCoV type II, while isolates of both genotypes were simultaneously detected in 53 samples.
The genes encoding the glycoprotein VP7, the VP8* trypsin-cleavage product of the protein VP4, a fragment of the protein VP6 associated with subgroup (SG) specificity, and the enterotoxin NSP4 of rotavirus strains identified in diarrheic fecal samples of rabbits in Italy were sequenced. The Italian lapine rotavirus (LRV) strains possessed a G3 VP7, SG I VP6, and KUN-like NSP4, a gene constellation typical of LRVs. One LRV strain (30/96), isolated in 1996, shared the closest amino acid (aa) identity (87-96%) with the P[14] genotype, composed of human and LRV strains. Conversely, three LRV strains (160/01, 229/01, and 308/01), identified in 2001, were highly identical (90-95%) among each other, but showed low aa identity (34-77%) to the VP8* genotype-specific sequences of representative rotavirus strains of all remaining P genotypes. This report confirms the worldwide genetic constellations of LRVs and identifies a novel VP4 genotype in rabbits, tentatively proposed as genotype P[22].
The sequence of the S gene of a field canine coronavirus (CCoV), strain Elmo/02, revealed low nucleotide (61%) and amino acid (54%) identity to reference CCoV strains. The highest correlation (77% nt and 81.7% aa) was found with feline coronavirus type I. A PCR assay for the S gene of strain Elmo/02 detected analogous CCoVs of different geographic origin, all which exhibited at least 92-96% nucleotide identity to each other and to strain Elmo/02. The evident genetic divergence between the reference CCoV strains and the newly identified Elmo/02-like CCoVs strongly suggests that a novel genotype of CCoV is widespread in the dog population.
The genetic diversity of 16 canine coronavirus (CCoV) samples is described. Samples were obtained from pups infected naturally living in different areas. Sequence data were obtained from the M gene and pol1a and pol1b regions. The phylogenetic relationships among these sequences and sequences published previously were determined. The canine samples segregated in two separate clusters. Samples of the first cluster were intermingled with reference strains of CCoV genotype and therefore could be assigned to this genotype. The second cluster segregated separately from CCoV and feline coronavirus genotypes and therefore these samples may represent genetic outliers. The reliability of the classification results was confirmed by repeating the phylogenetic analysis with nucleotide and amino acid sequences from multiple genomic regions.
The authors report the sequence analysis of the capsid protein-encoding gene (ORF2) of a calicivirus strain recently isolated in Italy from a pup with enteritis. Sequence comparison and phylogenetic analysis revealed that the isolate is highly similar to field and reference feline caliciviruses (FCVs). The isolation of a feline-like calicivirus and the results of recent serological investigations suggest that FCV infection frequently occurs in dogs, but the consequences of this interspecies infection in the canine host are still unknown.
Comparative sequence analysis of the PCR products of the M gene and fragments of the pol1a and pol1b genes of canine coronavirus (CCoV) have demonstrated that two separate clusters of CCoV are present in dogs. This note describes a PCR assay to identify atypical CCoV strains with nucleotide substitutions in the M gene. A total of 177 faecal samples from dogs CCoV positive previously with the PCR assay were analysed. Sixty-two of the 177 samples were amplified with the PCR described in the present study and were thus considered atypical CCoVs. The specificity of the PCR typing assay was confirmed by sequence analysis of the PCR products.
Two isolates of canine parvovirus (CPV) were obtained from dogs affected with severe haemorrhagic diarrhoea. Type 2b antigenic specificity was predicted by both antigenic analysis with monoclonal antibodies and PCR characterization with type-specific primers. Nevertheless, sequence analysis of the capsid protein-encoding gene revealed two amino acid changes. One of the changes affected position 426 (Asp to Glu), in a major antigenic site of the viral capsid, determining the replacement of a residue unique to CPV type 2b. The failure of established typing methods to distinguish this antigenic variant was overcome by the development of an RFLP assay.