A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA.

Aims: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples.

Methods And Results: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed.

Specific identification of feline panleukopenia virus and its rapid differentiation from canine parvoviruses using minor groove binder probes.

Taking into account reports of the isolation of canine parvoviruses (CPVs) from faecal samples of cats, we developed a real-time PCR assay, based on minor groove binder (MGB) probe technology, for rapid discrimination between true feline panleukopenia viruses (FPLVs) from CPVs. The assay takes advantage of a single nucleotide polymorphism at position 3753 of the viral genome (corresponding to residue 323 of the capsid VP2 protein) and of the ability of MGB probes to bind specifically only to perfectly complementary sequences. The FPV/CPV assay was proven to be highly specific, sensitive and reproducible and correlated well with a TaqMan assay able to recognise canine as well as feline parvoviruses.

Occurrence of canine parvovirus type 2c in the United States.

Canine parvovirus (CPV) type 2 (CPV-2) emerged around 1978 as a major pathogen of dogs worldwide. In the mid-1980s, the original CPV-2 had evolved and was completely replaced by 2 variants, CPV-2a and CPV-2b. In 2000, a new variant of CPV (named CPV-2c) was detected in Italy and now cocirculates with types 2a and 2b in that country.

Use of real-time RT-PCR as a rapid molecular approach for differentiation of field and vaccine strains of bluetongue virus serotypes 2 and 9.

Since 2000 severe, long-lasting epidemics of bluetongue virus (BTV) have been described in Italy, caused by BTV serotypes 2, 4, 9 and 16. Vaccination programs have been applied extensively to control the infection, in spite of concerns about the potential dissemination of attenuated vaccine strains of BTV in susceptible animals. Accordingly, rapid and reliable differentiation between vaccine and field strains is paramount in routine diagnosis of BTV to evaluate the extent of this phenomenon.

Detection of infectious canine parvovirus type 2 by mRNA real-time RT-PCR.

A TaqMan real-time RT-PCR assay was developed for detection of RNA transcripts produced by replicating CPV-2. A pair of primers and a TaqMan probe targeting the spliced NS2 mRNA were designed. A synthetic DNA fragment was constructed to mimic the spliced NS2 mRNA by PCR-based gene assembly and was used for generation of standard RNAs.

Severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season.

A severe outbreak of enteric and respiratory disease associated with bovine coronavirus (BCoV) infection is described. The outbreak occurred in a dairy herd of southern Italy in the first decade of September 2006, when summer temperatures were still recorded, affecting calves, heifers and adult cows, with a marked decrease in milk production. By virus isolation and RT-PCR targeting the S gene, BCoV was identified as the etiological agent of the outbreak, whereas bacteriological, parasitological and toxicological investigations failed to detect other causes of disease.

Genetic heterogeneity in the VP7 of group C rotaviruses.

Evidence for a possible zoonotic role of group C rotaviruses (GCRVs) has been recently provided. To gain information on the genetic relationships between human and animal GCRVs, we sequenced the VP7 gene of 10 porcine strains detected during a large surveillance study from different outbreaks of gastroenteritis in piglets. Four GCRV strains were genetically related to the prototype GCRV porcine Cowden strain.

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR.

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood).

Prevalence of group C rotaviruses in weaning and post-weaning pigs with enteritis.

Diarrheic fecal specimens collected from porcine herds were screened for the presence of group C rotaviruses using a reverse transcription-polymerase chain reaction (RT-PCR) assay. A total of 188 samples were tested and 54 were positive. When compiled these data with diagnostic results on group A rotaviruses and enteric caliciviruses we found that all but 5 group C rotavirus positive samples contained at least one additional virus.

Indomethacin has a potent antiviral activity against SARS coronavirus.

Unlabelled: Severe acute respiratory syndrome (SARS) is a newly emerging, highly transmissible and fatal disease caused by a previously unknown coronavirus (SARS-CoV). Existing in non-identified animal reservoirs, SARS-CoV continues to represent a threat to humans because there is no effective specific antiviral therapy for coronavirus infections.

Objectives: Starting from the observation that cyclopentenone cyclooxygenase (COX) metabolites are active against several RNA viruses, we investigated the effect of the COX inhibitor indomethacin on coronavirus replication.

Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks.

CDV is a highly contagious viral pathogen causing a lethal systemic disease in dogs and other carnivores. Several lineages or genotypes of CDV exist that are variously distributed throughout several continents. Legal or uncontrolled trading of animals may modify the epidemiology of CDV, introducing novel strains in CDV-naïve areas or accounting for the resurgence of CDV in areas where vaccine prophylaxis was effective and successful to control the disease.

Molecular characterisation of the virulent canine coronavirus CB/05 strain.

This paper characterises a virulent strain (CB/05) of canine coronavirus (CCoV) isolated from the internal organs of pups that had died of a systemic disease without evidence of other common canine pathogens. High viral RNA titres were detected in the internal organs by a real-time RT-PCR assay specific for CCoV type II. Sequence analysis of the 3' end (8.

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.

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.

Infectious canine hepatitis: an "old" disease reemerging in Italy.

Four outbreaks of infectious canine hepatitis (ICH) occurring in Italy between 2001 and 2006 are reported. Three outbreaks were observed in animal shelters of southern Italy, whereas a fourth outbreak involved two purebred pups imported from Hungary few days before the onset of clinical symptoms. In all outbreaks canine adenovirus type 1 (CAV-1) was identified by virus isolation and PCR.

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.

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.

First detection of canine parvovirus type 2c in pups with haemorrhagic enteritis in Spain.

Canine parvovirus type 2 (CPV-2), the aetiological agent of haemorrhagic enteritis in dogs, includes three antigenic variants, types 2a, 2b and 2c. CPV-2c has been detected initially in Italy and subsequently in Vietnam. We report the first identification of this novel antigenic variant in Spain, where it caused an outbreak of fatal enteritis in basset hound pups in association with canine coronavirus type I and type II.

Identification of group A porcine rotavirus strains bearing a novel VP4 (P) Genotype in Italian swine herds.

The VP4 gene of a G5 Italian porcine rotavirus strain, 344/04-1, was nontypeable by PCR genotyping. The amino acid sequence of the full-length VP4 protein had low identity ( View Article and Find Full Text PDF

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.

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.

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.

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.

Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy.

Canine distemper virus (CDV) is a highly contagious viral pathogen causing lethal disease in dogs and other mammalians. A high degree of genetic variation is found between recent CDV strains and the old CDV isolates used in the vaccines and such genetic variation is regarded as a possible cause of the increasing number of CDV-related diseases in dogs. The H gene shows the greatest extent of genetic variation that allows for distinction of various lineages, according to a geographical pattern of distribution and irrespective of the species of identification.

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.