Biomedical subjects

Nicola Decaro

Publications and source records attributed to Nicola Decaro.

At least 19 recordsLinked to original sources

PubMed2006

Serological and molecular evidence that canine respiratory coronavirus is circulating in Italy.

Canine respiratory coronavirus (CRCoV) is a group II coronavirus that was firstly identified in lung samples of dogs with canine infectious respiratory disease (CIRD) in UK in 2003. We report for the first time the identification of CRCoV in Italy, together with serological evidence that the virus has been circulating in the Italian dog population as from 2005. Serological investigations on 216 dog sera, carried out by an ELISA test using the strictly related bovine coronavirus (BCoV) as antigen, revealed an overall CRCoV seroprevalence of 32.06% in the last 2 years. RT-PCR targeting the S-gene of CRCoV was carried out on 109 lung samples collected from carcasses of dogs submitted for diagnostic investigations. Positive results were obtained from the lungs of a dog of the Apulia region that was co-infected with canine parvovirus type 2. Sequence analysis of the S-gene fragment amplified by RT-PCR (595bp) showed similarity to group II coronaviruses, with the highest nucleotide identity (98%) to the only CRCoV strain currently available in the GenBank database (strain T101). The results of the present study show that CRCoV is present also in continental Europe, although further studies are required to determine the real pathogenic potential of the virus.

Animals↗

PubMed2006

An outbreak of equine influenza virus in vaccinated horses in Italy is due to an H3N8 strain closely related to recent North American representatives of the Florida sub-lineage.

In December 2005, equine influenza virus infection was confirmed as the cause of clinical respiratory disease in vaccinated horses in Apulia, Italy. The infected horses had been vaccinated with a vaccine that contained strains representatives from both the European (A/eq/Suffolk/89) and American (A/eq/Newmarket/1/93) H3N8 influenza virus lineages, and the H7N7 strain A/eq/Praga/56. Genetic characterization of the hemagglutinin (HA) and neuraminidase (NA) genes of the virus from the outbreak, indicated that the isolate (A/eq/Bari/2005) was an H3N8 strain closely related to recent representatives (Kentucky/5/02-like) of the American sub-lineage Florida, that was introduced in Italy through movement of infected horses from a large outbreak described in 2003 in United Kingdom. Strain A/eq/Bari/2005 displayed 9 amino acid changes in the HA1 subunit protein with respect to the reference American strain A/eq/Newmarket/1/93 contained in the vaccine. Four changes were localized in the antigenic regions C-D and likely accounted for the vaccine failure.

Amino Acid Sequence↗

PubMed2006

Tissue distribution of the antigenic variants of canine parvovirus type 2 in dogs.

Twelve dogs dead as consequence of natural infection caused by canine parvovirus (CPV) type 2a (n=4), type 2b (n=4) or type 2c (n=4) were investigated for determining the viral DNA loads in different tissue samples. By means of a real-time PCR assay, CPV DNA was detected in all tissues examined, with the highest titres observed in the lymphoid tissue and the lowest loads in the urinary tract. Surprisingly, the nervous tissue was found to contain considerable amounts of CPV nucleic acid. Similar patterns of tissue distribution were observed in all the examined dogs irrespective of the antigenic variant causing the disease.

Animals↗

PubMed2006

Intravaginal administration of an inactivated vaccine prevents lesions induced by caprine herpesvirus-1 in goats.

To evaluate the efficacy of mucosal vaccination with a beta-propiolactone inactivated caprine herpesvirus-1 (CpHV-1) vaccine, goats received vaginal administrations of two 7-day cycles at 2 weeks intervals. Seven days after the end of the second cycle, goats were challenged intravaginally with 4 ml of virulent BA.1 strain of CpHV-1. Vaccinated goats shed challenge virus for 8 days but, in comparison with control unvaccinated-challenged goats, were significantly protected from the classical clinical signs of genital lesions. Even without adjuvants, prolonged mucosal vaccination induced production of secretory IgA and provided significant clinical protection.

Administration, Intravaginal↗

PubMed2006

Occurrence of severe gastroenteritis in pups after canine parvovirus vaccine administration: a clinical and laboratory diagnostic dilemma.

A total of 29 faecal samples collected from dogs with diarrhoea following canine parvovirus (CPV) vaccination were tested by minor groove binder (MGB) probe assays for discrimination between CPV vaccine and field strains and by diagnostic tests for detection of other canine pathogens. Fifteen samples tested positive only for CPV field strains; however, both vaccine and field strains were detected in three samples. Eleven samples were found to contain only the vaccine strain, although eight of them tested positive for other pathogens of dogs. Only three samples were found to contain the vaccine strain without evidence of canine pathogens. The present study confirms that most cases of parvovirus-like disease occurring shortly after vaccination are related to infection with field strains of canine parvovirus type 2 (CPV-2) rather than to reversion to virulence of the modified live virus contained in the vaccine.

Animals↗

PubMed2006

Diagnostic tools based on minor groove binder probe technology for rapid identification of vaccinal and field strains of canine parvovirus type 2b.

TaqMan-based diagnostic tests have been developed for the identification of canine parvovirus type 2 (CPV-2) strains in the faeces of dogs with diarrhoea, including a minor groove binder (MGB) probe assay for identification of type 2-based vaccines and field strains (types 2a, 2b and 2c). Since type 2b vaccines have been licensed recently in Europe, two novel MGB assays were developed for discrimination between type 2b vaccines and field strains of CPV. Such assays have been found to be highly sensitive, specific and reproducible, allowing for simultaneous detection of type 2b vaccinal and field strains present in the same specimens. These new assays will help resolution of the diagnostic problems related to the detection of a type 2b strain in the faeces of dogs shortly after the administration of a type 2b vaccine.

Analysis of Variance↗

PubMed2006

Detection of canine distemper virus in dogs by real-time RT-PCR.

Canine distemper virus is the etiological agent of a severe disease in dogs and many other carnivores. Clinical diagnosis of canine distemper is difficult due to the broad spectrum of signs that may be confounded with other respiratory and enteric diseases of dogs. Accordingly, a laboratory confirmation is required for suspected cases. In this study a real-time RT-PCR assay was developed for detection and quantitation of canine distemper virus. The assay exhibited high specificity as all the negative controls (no-template-controls and samples from healthy sero-negative dogs) and other canine pathogens were not misdetected. Up to 1 x 10(2) copies of RNA were detected by the TaqMan assay, thus revealing a high sensitivity. Quantitative TaqMan was validated on clinical samples, including various tissues and organs collected from dogs naturally infected by canine distemper virus. Urines, tonsil, conjunctival swabs and whole blood were found to contain high virus loads and therefore proved to be suitable targets for detection of canine distemper virus RNA.

Animal Structures↗

PubMed2006

A minor groove binder probe real-time PCR assay for discrimination between type 2-based vaccines and field strains of canine parvovirus.

A minor groove binder (MGB) probe assay was developed to discriminate between type 2-based vaccines and field strains of canine parvovirus (CPV). Considering that most of the CPV vaccines contain the old type 2, no longer circulating in canine population, two MGB probes specific for CPV-2 and the antigenic variants (types 2a, 2b and 2c), respectively, were labeled with different fluorophores. The MGB probe assay was able to discriminate correctly between the old type and the variants, with a detection limit of 10(1) DNA copies and a good reproducibility. Quantitation of the viral DNA loads was accurate, as demonstrated by comparing the CPV DNA titres to those calculated by means of the TaqMan assay recognising all CPV types. This assay will ensure resolution of most diagnostic problems in dogs showing CPV disease shortly after CPV vaccination, although it does not discriminate between field strains and type 2b-based vaccines, recently licensed to market in some countries.

Animals↗

PubMed2006

Evolution of CPV-2 and implication for antigenic/genetic characterization.

A few amino acid differences in the viral protein VP2 account for important antigenic and biological changes among feline parvovirus (FPV), canine parvovirus (CPV-2) and CPV-2 variants 2a and 2b. Several pieces of evidence suggest that CPV-2 is still evolving as additional amino acid changes occurred within the main antigenic regions of CPV-2 capsid, altering the antigenic profile of the virus and stressing the need for implementing the diagnostic assays.

Animals↗

PubMed2006

Canine coronavirus highly pathogenic for dogs.

Canine coronavirus (CCoV) is usually responsible for mild, self-limiting infections restricted to the enteric tract. We report an outbreak of fatal disease in puppies caused by a pathogenic variant of CCoV that was isolated from organs with severe lesions.

Animals↗

PubMed2005

Characterisation of the canine parvovirus type 2 variants using minor groove binder probe technology.

Two minor groove binder (MGB) probe assays were developed for rapid identification and quantitation of the canine parvovirus type 2 (CPV-2) variants in the faecal samples of dogs. The MGB probe assays using type-specific probes labeled with different fluorophores (FAM and VIC) were able to detect the single nucleotide polymorphisms existing between types 2a/2b and 2b/2c. Both the MGB probe assays were found to be highly specific, sensitive and reproducible, ensuring a precise quantitation of the CPV-2 DNA, as confirmed by comparing the obtained viral DNA loads to those calculated using a real-time TaqMan-based PCR assay established previously. A total of 414 faecal specimens collected from diarrhoeic dogs and tested positive to CPV-2 were analysed by the MGB probe assays and by conventional methods (haemagglutination inhibition with MAbs, PCR-RFLP and sequence analysis) for prediction of the CPV-2 antigen specificity, with an overall agreement of 100%. These results make the established MGB probe assays an attractive tool for direct and rapid characterisation of type 2a, 2b and 2c CPVs.

Amino Acid Substitution↗

PubMed2005

Detection of equine herpesvirus type 1 by real time PCR.

A real-time PCR assay was developed for detection and quantitation of equid herpesvirus type 1 (EHV-1). The sensitivity of the assay was compared with an established nested-PCR (n-PCR). The real-time PCR detected 1 copy of target DNA, with a sensitivity 1 log higher than gel-based n-PCR. The assay was able to detect specifically EHV-1 DNA in equine tissue samples and there was no cross-amplification of other horse herpesviruses. Real-time PCR was applied to determine EHV-1 load in tissue samples from equine aborted fetuses. The high sensitivity and reproducibility of the EHV-1-specific fluorogenic PCR assay, combined with the wide dynamic range and the high throughput, make this method suitable for diagnostic and research applications.

Aborted Fetus↗

PubMed2005

Relationships among porcine and human P[6] rotaviruses: evidence that the different human P[6] lineages have originated from multiple interspecies transmission events.

Porcine rotavirus strains (PoRVs) bearing human-like VP4 P[6] gene alleles were identified. Genetic characterization with either PCR genotyping or sequence analysis allowed to determine the VP7 specificity of the PoRVs as G3, G4, G5 and G9, and the VP6 as genogroup I, that is predictive of a subgroup I specificity. Sequence analysis of the VP8* trypsin-cleavage product of VP4 allowed PoRVs to be characterized further into genetic lineages within the P[6] genotype. Unexpectedly, the strains displayed significantly higher similarity (up to 94.6% and 92.5% at aa and nt level, respectively) to human M37-like P[6] strains (lineage I), serologically classifiable as P2A, or to the atypical Hungarian P[6] human strains (HRVs), designated as lineage V (up to 97.0% aa and 96.1% nt), than to the porcine P[6] strain Gottfried, lineage II (<85.1% aa and 82.2 nt), which is serologically classified as P2B. Interestingly, no P[6] PoRV resembling the original prototype porcine strain, Gottfried, was detected, while Japanase P[6] PoRV clustered with the atypical Japanase G1 human strain AU19. By analysis of the 10th and 11th genome segments, all the strains revealed a NSP4B genogroup (Wa-like) and a NSP5/6 gene of porcine origin. These findings strongly suggest interspecies transmission of rotavirus strains and/or genes, and may indicate the occurrence of at least 3 separate rotavirus transmission events between pigs and humans, providing convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.

Amino Acid Sequence↗

PubMed2005

Analysis of antibody response in goats to caprine herpesvirus 1.

Serum samples of goats experiencing natural and experimental infections and/or reactivation of caprine herpesvirus 1 (CpHV.1) were analysed with neutralization and Western blotting (WB) tests. WB immunological patterns resulted differently and related to neutralizing titers. In serum samples having neutralizing titer 1:2-1:4, antibodies to two proteins of Mw of 150 and 34 kDa were present. Antibodies against several proteins, two of those being characterized by monoclonal antibodies as gB and gC, were visualized by WB in sera having titer > or = 1:8. The neutralizing antibody titers and the pattern of antibody reactivity were hypothesized to modulate the reactivation and re-excretion process of CpHV.1.

Animals↗

PubMed2005

Maternally-derived antibodies in pups and protection from canine parvovirus infection.

The interaction between maternally-derived antibodies (MDA) and canine parvovirus (CPV) infection was evaluated in five groups of pups with a wide range of haemagglutination inhibiting (HI) titres of MDA (from < 10 to 320). The pups were inoculated with a field CPV strain and monitored daily to evaluate their clinical condition and viral shedding in the faeces. Serum samples were collected weekly to evaluate antibody response. Clinical signs were observed in dogs with HI titres up to 80. Active CPV replication was demonstrated in dogs with HI titres up to 160, although slightly delayed, at lower titres and for a shorter period compared to seronegative dogs. The successful infection of dogs with HI titres of 80 and 160 was also confirmed by seroconversion, evaluated at day 14 postinfection. These findings are in contrast with the MDA titre (HI > or = 80) usually considered fully protective for CPV infection, and suggest the need for revision of current vaccination programmes for pups.

Animals↗

PubMed2005

Virological and molecular characterization of a mammalian orthoreovirus type 3 strain isolated from a dog in Italy.

A mammalian orthoreovirus (MRV) strain was isolated from a pup with fatal diarrhea, which had a concurrent infection by canine parvovirus type 2. The reovirus isolate showed an atypical hemagglutination pattern and a retarded electrophoretic mobility of the S1 segment, which is characteristic of MRV type 3 (MRV-3). Assignment of the isolated virus to MRV-3 was confirmed by type-specific RT-PCR assays, targeting the S1 gene, and by subsequent sequence analysis of the PCR product. By phylogeny based on the S1 gene of several MRVs, the isolate fell into lineage E, along with the murine strain T3C9/61 and the bovine strains T3C18/61 and T3C31/59. Conversely, L1 sequences were found to segregate regardless of the viral type. A total of 110 fecal samples, 56 nasal and 31 ocular swabs from dogs with diarrhea or nasal/ocular discharge were tested by a nested-PCR assay specific for reoviruses, and no sample was found to contain MRV RNA, a finding that is apparently in contrast with the seroprevalence (25.77%) observed in dogs.

Animals↗

PubMed2005

Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs.

Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 10(1) to 10(8) copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.

Animals↗

PubMed2005

Canine parvovirus infection: which diagnostic test for virus?

Five laboratory tests for diagnosis of canine parvovirus type 2 (CPV-2) infection were employed to test 89 faecal samples collected from dogs with diarrhoea. The tests analysed were immunochromatography (IC), haemagglutination (HA), virus isolation (VI), conventional and real-time PCR. IC, HA, VI and conventional or real-time PCR were able, respectively, to detect CPV-2 antigen or nucleic acid in 41, 50, 54, 68 and 73 of the samples. The best correlation was found between conventional and real-time PCR, with an overall agreement of 94.38%. Sixty-eight samples that tested positive by HA, VI or conventional PCR were subjected to antigenic and/or genetic analyses of the CPV-2 strains by monoclonal antibody (MAb), restriction fragment-length polymorphism (RFLP) and/or sequence analyses. In sum, out of the 68 strains analysed, 26 were characterised as CPV-2a, 18 as CPV-2b and 24 as a CPV-2 Glu-426 mutant recently identified in Italy.

Animals↗