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We monitored the severe acute respiratory syndrome coronavirus 2 antibody response in seven dogs and two cats by using two multispecies ELISA tests, plaque reduction neutralisation test and virus neutralization. SARS‐CoV‐2 neutralizing antibodies in pets persisted up to 10 months since the first positive testing, thus replicating observations in COVID‐19 human patients. [ABSTRACT FROM AUTHOR]

Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. The unpredictable and variable course of CDV-related disease may hamper correct diagnosis of infection and makes it crucial the collection of samples suitable for laboratory confirmation. In the present study we were able to follow the disease in two dogs infected naturally, collecting different biological matrices during the entire period of infection. By real time RT-PCR, viral RNA was detected and quantified, suggesting that urine and rectal swabs would be useful for ante-mortem diagnosis of distemper in dogs, regardless of the clinical stage and form of the illness. [ABSTRACT FROM AUTHOR]

Abstract: 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 1×102 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. [Copyright &y& Elsevier]

Abstract: 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 101 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 102 to 109 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. [Copyright &y& Elsevier]

An enzyme-linked immunosorbent assay (Elisa), using as antigen canine coronavirus-infected CrFK cell supernatant, was developed to detect antibodies against canine coronavirus (CCoV). Out of a total of 109 dog serum samples, 80 which were positive by routine virus neutralisation test were also Elisa positive. Seventeen samples which were negative by the virus neutralisation test, were positive by Elisa and by the confirmatory Western blotting test. The Elisa was substantially more sensitive than the virus neutralisation test in detecting antibodies to CCoV and may be used as an alternative technique to virus neutralisation. [Copyright &y& Elsevier]

The epidemiological role of domestic animals in the spread and transmission of SARS-CoV-2 to humans has been investigated in recent reports, but some aspects need to be further clarified. To date, only in rare cases have dogs and cats living with COVID-19 patients been found to harbour SARS-CoV-2, with no evidence of pet-to-human transmission. The aim of the present study was to verify whether dogs and cats act as passive mechanical carriers of SARS-CoV-2 when they live in close contact with COVID-19 patients. Cutaneous and interdigital swabs collected from 48 dogs and 15 cats owned by COVID-19 patients were tested for SARS-CoV-2 by qRT-PCR. The time elapsed between owner swab positivity and sample collection from pets ranged from 1 to 72 days, with a median time of 23 days for dogs and 39 days for cats. All samples tested negative, suggesting that pets do not passively carry SARS-CoV-2 on their hair and pads, and thus they likely do not play an important role in the virus transmission to humans. This data may contribute to confirming that the direct contact with the hair and pads of pets does not represent a route for the transmission of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Canine coronavirus types I (CCoV-I) and II (CCoV-II) are usually responsible for mild enteritis in dogs. While the CCoV-II genome has been completely sequenced, to date there are no complete genomic sequence data available publicly for CCoV-I. Thus, the aim of the present study was to analyze the full-length genome of a CCoV-I prototype strain that had been recovered from a dog with diarrhea in Italy. CCoV-I strain 23/03 has a genome of 30,000 nucleotides, excluding the 3′ poly(A) tail, displaying the typical Alphacoronavirus-1 organization and the highest genetic relatedness to CCoV-II. However, two distinct features were observed in the CCoV-I genome: (i) the presence of an additional ORF between the spike (S) protein gene and ORF3a; (ii) the diversity of the S protein, which is more closely related to that of feline coronavirus type I and presents a furin cleavage site. The present study may contribute to a better understanding of the Alphacoronavirus-1 evolutionary pattern and may be paradigmatic of how coronaviruses evolve through gene losses, acquisition and exchanges among different members. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Canine circovirus (CanineCV) is a canine virus, whose pathogenetic role is still uncertain. Based on recent data suggesting its role as entheropathogen, a case-control study was conducted between 2013 and 2016 to investigate the association of CanineCV with gastroenteritis in dogs, alone or in combination with other viral pathogens, including canine parvovirus (CPV), canine coronavirus (CCoV) and canine distemper virus (CDV). A total of 219 dogs suffering from acute gastroenteritis disorders and 67 controls randomly recruited among healthy dogs or patients presenting without enteric signs were screened by a panel of real-time (RT-)PCR assays for CanineCV, CPV, CCoV and CDV. A high prevalence of viral infections was detected in dogs with gastroenteritis (77.16%), with CPV representing the most frequently detected enteropathogen, followed by CanineCV and CCoV. While CPV and CCoV infections displayed a strong association with occurrence of acute gastroenteritis ( p < 0.00001), detection of CanineCV in control dogs (28.35%) occurred with prevalence comparable to that of clinical cases (32.42%), so that its correlation with gastrointestinal disease was not statistically supported ( p = 0.530988). Different from the clinical cases, where co-infections were frequently observed, all positive samples from the control group contained single infections. Noteworthy, a significant association was calculated between co-infections with CanineCV and occurrence of acute gastroenteritis ( p < 0.00001). This study supports the role of CanineCV as a co-pathogen in the development of gastrointestinal disease, mainly acting in synergism with other enteric viruses. [ABSTRACT FROM AUTHOR]

A molecular survey for traditional and emerging pathogens associated with canine infectious respiratory disease (CIRD) was conducted in Italy between 2011 and 2013 on a total of 138 dogs, including 78 early acute clinically ill CIRD animals, 22 non-clinical but exposed to clinically ill CIRD dogs and 38 CIRD convalescent dogs. The results showed that canine parainfluenza virus (CPIV) was the most commonly detected CIRD pathogen, followed by canine respiratory coronavirus (CRCoV), Bordetella bronchiseptica , Mycoplasma cynos , Mycoplasma canis and canine pneumovirus (CnPnV). Some classical CIRD agents, such as canine adenoviruses, canine distemper virus and canid herpesvirus 1, were not detected at all, as were not other emerging respiratory viruses (canine influenza virus, canine hepacivirus) and bacteria ( Streptococcus equi subsp. zooepidemicus ). Most severe forms of respiratory disease were observed in the presence of CPIV, CRCoV and M. cynos alone or in combination with other pathogens, whereas single CnPnV or M. canis infections were detected in dogs with no or very mild respiratory signs. Interestingly, only the association of M. cynos (alone or in combination with either CRCoV or M. canis ) with severe clinical forms was statistically significant. The study, while confirming CPIV as the main responsible for CIRD occurrence, highlights the increasing role of recently discovered viruses, such as CRCoV and CnPnV, for which effective vaccines are not available in the market. [ABSTRACT FROM AUTHOR]

Abstract: A pantropic canine coronavirus (CCoV) strain (CB/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. We report the clinical, virological and serological findings in dogs experimentally infected with strain CB/05. The dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral RNA with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. Leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. Considering the severity of the CB/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8–9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. At postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for CCoV by real-time RT-PCR and virus isolation on cell cultures. All pups seroconverted for CCoV, as shown by the high optical density values and antibody titres detected by ELISA and virusneutralisation tests, respectively. The present study confirms that strain CB/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions. [Copyright &y& Elsevier]

Abstract: 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≥80) usually considered fully protective for CPV infection, and suggest the need for revision of current vaccination programmes for pups. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

An epidemiological survey for canine parvovirus (CPV) was conducted by collecting 615 faecal samples from dogs with diarrhoea in different European countries. Molecular methods showed that CPV-2a was predominant in most countries, followed by CPV-2c and CPV-2b, whereas 30 strains were not characterised. By sequence analysis of the full-length VP2 gene, 20 of these viruses were characterised as CPV-2c mutants having the synonymous mutation A4061G in the probe-binding region that prevented correct strain characterisation. A real-time polymerase chain reaction (PCR) assay using a minor groove binder probe was able to recognise both mutant and classical CPV-2c strains. These results indicate that the emergence of CPVs with mutations affecting the oligonucleotide-binding region needs a continuous update of molecular diagnostic tools in order to detect efficiently those emerging strains. [ABSTRACT FROM AUTHOR]