Search

Four canine coronavirus type II (CCoV-II) strains were identified in the guts and internal organs of pups which had died of acute gastroenteritis. The CCoV-II strains were strictly related to porcine transmissible gastroenteritis virus (TGEV) in the N-terminal domain of the spike protein, whereas in the other parts of the genome, a higher genetic relatedness to recent CCoV-II isolates was observed. Experimental infection of dogs with a TGEV-like isolate induced mild gastroenteritis without any systemic involvement. By virus neutralization tests, antigenic differences between reference and TGEV-like CCoVs were found. Our data support the potential recombinant origin of the TGEV-like CCoVs. Copyright © 2009, American Society for Microbiology. All Rights Reserved., This work was supported by grants from the Italian Ministry of Health, Ricerca finalizzata 2008, project Mammalian Coronaviruses: Molecular Epidemiology, Vaccine Development and Implications for Animal and Human Health (Nicola Decaro), and from the Italian Ministry of Education, University and Research, PRIN 2006, project Infezione del Cane da Coronavirus Pantropico: Aspetti Epidemiologici, Patogenetici e Molecolari (Canio Buonavoglia).

Malignant catarrhal fever (MCF) is a fatal, systemic disease of cattle and other domestic and wild ruminants that, in Europe, is caused by Ovine herpesvirus 2 (OvHV-2). American bison ( Bison bison) are highly susceptible to the disease. An adult American bison, housed in a zoo in southern Italy in close cohabitation with a group of domestic sheep ( Ovis aries aries) displayed clinical signs that resembled the acute form of MCF. By real-time polymerase chain reaction, OvHV-2 DNA was detected intravitam in blood, in nasal and ocular swabs, and postmortem in tissue samples of the bison. By indirect fluorescent antibody test, high MCF antibody titers were found in the bison serum. Ovine herpesvirus 2 DNA and antibodies were also found in blood samples from the domestic sheep, thus suggesting a potential role of these animals as a source of the infection. To the authors' knowledge, this is the first MCF case in captive ruminants in Italy and the second confirmed case in captive bison of European zoos.

Caprine herpesvirus 1 (CpHV-1) infection in goats induces genital vesicular-ulcerative lesions that strictly resemble those produced by human herpesvirus 2 in humans. In previous studies, the potent inhibition of CpHV-1 by cidofovir was demonstrated. Cidofovir antiherpetic activity was evaluated in goats infected experimentally by the vaginal route with CpHV-1 and then treated locally at different times after infection. The administration of 1% cidofovir cream onto vaginal mucosa was able to prevent the onset of genital lesions and to decrease significantly the titers of the virus shed by the infected animals, notably in the groups treated shortly after infection (24 and 48 h). The efficacy of cidofovir against caprine herpesvirus infection was higher when the treatment was started shortly after infection than when lesions were already present and advanced. Herpesvirus genital infection of goats is a useful animal model to study the activity of antiviral drugs against human herpesvirus infections.

Thirty-nine parvovirus strains contained in faecal samples collected in Italy (n=34) and UK (n=5) from cats with feline panleukopenia were characterized at the molecular level. All viruses were proven to be true feline panleukopenia virus (FPLV) strains by a minor groove binder probe assay, which is able to discriminate between FPLV and the closely related canine parvovirus type 2. By using sequence analysis of the VP2 gene, it was found that the FPLV strains detected in Italy and UK were highly related to each other, with a nucleotide identity of 99.1–100 and 99.4–99.8 % among Italian and British strains, respectively, whereas the similarities between all the sequences analysed were 98.6–100 %. Eighty-eight variable positions were detected in the VP2 gene of the field and reference FPLV strains, most of which were singletons. Synonymous substitutions (n=57) predominated over non-synonymous substitutions (n=31), and the ratio between synonymous and non-synonymous substitutions (dN/dS) was 0.10, thus confirming that evolution of FPLV is driven by random genetic drift rather than by positive selection pressure. Some amino acid mutations in the VP2 protein affected sites that are thought to be responsible for antigenic and biological properties of the virus, but no clear patterns of segregation and genetic markers, were identified, confirming that FPLV is in evolutionary stasis.

First report of bovine anaplasmosis by Anaplasma centrale in Europe, molecular identification and phylogenetic analysis L. Ceci & N. Decaro & E. Lorusso & P. Paradies & G. Elia & V. Martella & C. Buonavoglia & G. Carelli Published online: 12 August 2008 # Springer Science + Business Media B.V. 2008

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of bovine coronavirus (BCoV) RNA in clinical samples is described. The assay is based on TaqMan technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the transmembrane-protein gene of BCoV. The BCoV real-time RT-PCR assay was able to detect the tested BCoV and BCoV-like viruses (canine respiratory coronavirus and bubaline coronavirus), whereas other common viral pathogens of cattle were not recognised by the established oligonucleotide set, thus showing that the test was specific for bovine-like CoVs. The detection limit of the assay was 20 BCoV RNA copies (1-log higher with respect to traditional gel-based RT-PCR) and the reproducibility was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. Two hundred and twenty clinical specimens (92 rectal, 82 nasal and 46 ocular swabs) were subjected to gel-based and real-time RT-PCR. By conventional amplification, 43 rectal, 54 nasal and 34 ocular samples tested positive, whereas the TaqMan assay was able to detect the BCoV nucleic acid in 49 rectal, 60 nasal and 37 ocular swabs. The rapidity and high throughput of the BCoV TaqMan assay makes this method a powerful tool for a sensitive and specific diagnosis of BCoV infection in cattle.

Vaccine-based prophylaxis has greatly helped to keep distemper disease under control. Notwithstanding, the incidence of canine distemper virus (CDV)-related disease in canine populations throughout the world seems to have increased in the past decades, and several episodes of CDV disease in vaccinated animals have been reported, with nation-wide proportions in some cases. Increasing surveillance should be pivotal to identify new CDV variants and to understand the dynamics of CDV epidemiology. In addition, it is important to evaluate whether the efficacy of the vaccine against these new strains may somehow be affected.

Canine adenoviruses (CAVs) and canine herpesvirus (CHV) are pathogens of dogs that have been known for several decades. The two distinct types of CAVs, type 1 and type 2, are responsible for infectious canine hepatitis and infectious tracheobronchitis, respectively. In the present article, the currently available literature on CAVs and CHV is reviewed, providing a meaningful update on the epidemiologic, pathogenetic, clinical, diagnostic, and prophylactic aspects of the infections caused by these important pathogens.

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.

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. In the present study, we report the development of two real-time RT-PCR assays able to recognise BTV serotypes 2 and 9, respectively, and we evaluated the use of the assays for discrimination between field and vaccine strains. A total of 65 samples collected in Italy from 2000 to 2006 and diagnosed as positive for either BTV-2 or -9 were analysed by the TaqMan assays. Both the assays were found to be highly sensitive and reproducible, ensuring correct serotype characterisation and prediction of the origin of the strains, as confirmed by characterisation using virus neutralisation and sequencing.

Despite vaccination, canine distemper virus (CDV) remains one of the important pathogen of dogs with worldwide distribution. Ribavirin (RIB) inhibits replication of measles virus (MV), a morbillivirus closely related to CDV, both in vitro and in vivo. In this report the antiviral activity of RIB against CDV in cell cultures was assessed. Quantitative real-time RT-PCR was used to measure viral RNA in VERO cells infected by CDV and to evaluate the inhibitory effects of RIB. RIB caused a dose- and time-dependent decrease in accumulation of CDV RNA when added after virus adsorption. RIB was highly effective in preventing CDV replication at low concentrations with 50% virus-inhibitory concentrations ranging from 0.02 to 0.05 mM. Such low values were comparable to values displayed by highly susceptible strains of MV. In addition, CDV was passaged sequentially in VERO cell monolayers in the presence of RIB to trigger viral extinction. The virus was no longer detected after three passages, suggesting that error catastrophe is one of the modes of action of RIB against CDV. These findings suggest RIB as a promising tool for the therapy of CD in dogs.

Evidence in several species, including dogs, has been collected demonstrating that the brain hemispheres modulate the immune system in an asymmetrical way. To study the interactions between immune response and lateralization, three groups of mixed breed dogs were selected on the basis of their performance in a paw preference test involving removal of a piece of sticky tape from the snout. The expression of interleukin-2 (IL-2) and interleukin-6 (IL-6) genes was measured in left-pawed, right-pawed and ambidextrous dogs before and after immunization treatment with a rabies vaccine. The results revealed a relationship between the mRNA expression of IL-2 and IL-6 genes and the direction of behavioural lateralization. Under basal conditions, IL-2 and IL-6 gene expression was higher in left-pawed dogs than in right-pawed and ambidextrous dogs. After the vaccine administration, decreasing levels of IL-2 and IL-6 gene expression were observed in left-pawed and right-pawed dogs, but not in ambidextrous dogs. These findings represent the first evidence that brain lateralization may influence the immune system in dogs by the modulation of mRNA gene expression of cytokines such as IL-2 and IL-6, which have been recognized as key immune-regulatory proteins.

Four outbreaks of bovine respiratory disease (BRD) associated with bovine Coronavirus (BCoV) infection in Italian cattle herds were reported. In 3 outbreaks, BRD was observed only in 2–3-month-old feedlot calves, whereas in the remaining outbreak, lactating cows, heifers, and calves were simultaneously affected. By using reverse transcription polymerase chain reaction (RT-PCR), BCoV RNA was detected in all outbreaks without evidence of concurrent viral pathogens (i.e., bovine respiratory syncytial virus, bovine herpesvirus type 1, bovine viral diarrhea virus, bovine parainfluenza virus). Common bacteria of cattle were recovered only from 2 outbreaks of BRD: Staphylococcus spp. and Proteus mirabilis (outbreak 1) and Mannheimia haemolytica (outbreak 4). A recently established real-time RT-PCR assay showed that viral RNA loads in nasal secretions ranged between 3.10 × 10 2 and 7.50 × 10 7 RNA copies/μl of template. Bovine Coronavirus was isolated from respiratory specimens from all outbreaks except outbreak 1, in which real-time RT-PCR found very low viral titers in nasal swabs.

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. The detection limit of the assay was 1 × 10 2 RNA copies and standard curve displayed a linear range from 1 × 10 2 to 1 × 10 9 copies and a good reproducibility. The assay was then applied to determine the mRNA loads in the tissues of dogs naturally infected by CPV-2. mRNA was detected in a variety of tissues, including the central nervous system.

Aims: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. Methods and Results: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16·7%) and mischaracterized three additional strains as MRSA (specificity of 98·45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92·3%). Fifteen MSSA strains displayed a β-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. Conclusions: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. Significance and Impact of the Study: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.

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. A hemi-nested PCR system was developed to genotype strains of the major CDV lineages, America-1, Europe, Asia-1, Asia-2 and Arctic. The assay was tested using a collection of 27 laboratory and vaccine strains and of 36 field CDV strains. Distinct lineages could be differentiated by specific primers targeted to the H gene. The method could be useful for molecular epidemiological studies of CDV, providing a tool for large-scale studies, and for the diagnosis of vaccine-related disease.

Caprine herpesvirus 1 (CpHV-1) is a virus able to cause genital infection leading to vulvovaginitis or balanoposthitis in adult goats. CpHV-1 shares several biological similarities with herpes simplex type 2 (HSV-2) infection in man, such as genital tropism, type and site of typical lesions and it might provide an animal model for studies on antiviral drugs for HSV-2 infection in man. In this view the efficacy of cidofovir (CDV) drug was tested in six goats intravaginally infected with BA.1 strain of CpHV-1. Three goats received an intravaginal application of 3 ml of a 1% CDV preparation at 4h post infection and then every 12 h for five consecutive days. Three goats were kept as untreated controls. The goats were daily examined for clinical evidence of the infection and viral shedding. CDV was able to protect against disease progression and inhibited the onset of the local lesions due to the CpHV-1 replication. Treated animals shed virus for a shorter period (3 days less) and at lower titres than the control animals. CpHV-1 infection in goats may represent an excellent animal model for the study of novel strategies for the treatment of primary genital HSV-2 infection in man.

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 (8.7 kb) of the genomic RNA of strain CB/05 revealed conserved structural as well as non-structural proteins, with the exception of a truncated form of non-structural protein 3b. The exceptional form was due to a 38-nucleotide deletion and a frame shift in ORF3b that introduced an early stop codon. By phylogenetic analysis of the structural proteins, the spike (S) protein was found to cluster with feline coronavirus type II strain 79-1683, whereas, the envelope (E), membrane (M) and nucleocapsid (N) proteins segregated together with the reference strain Purdue of transmissible gastroenteritis virus of swine.

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 (595 bp) 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.

The aim of this study was to screen for Brucella spp. buffalo embryos produced in- vitro, by using cumulus oocytes complexes (COCs) recovered from ovaries of slaughtered buffaloes naturally infected with Brucella spp. Ovaries were collected from 5 female pluriparous buffaloes slaughtered in a local abattoir. EDTA-blood samples and nasal swabs collected from each animal were used for Brucella spp. DNA detection by real-time PCR. Buffalo ovaries (n = 10) were transported to the laboratory and maintained strictly separated throughout laboratory processing. Recovered COCs were matured, fertilized and cultured in vitro until day 7. Some immature COCs, all uncleaved COCs, all blocked cleaved embryos (2 to 16 cells) and all transferable embryos (tight morulae and blastocysts) were separately analysed by real-time PCR assay. Brucella spp. DNA was detected in both blood and nasal mucus of all subjects, whereas no trace of DNA of Brucella spp. was found on either COCs or embryos. Currently, the infected or seropositive buffaloes have to be slaughtered for sanitary reasons. Interestingly, the results of this preliminary trial suggest a possible utilization of the COCs from the infected subjects of high genetic value to obtain safe embryos.

Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously-expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogues, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors.

Pantropic canine coronavirus (CCoV) was first detected in young dogs in Italy in 2005, but the complete genome sequence of this virus had not yet been determined. Here, we report the full-length genome sequence of the prototype strain CB/05, which showed that this virus is genetically similar to CCoV-IIa viruses.

Highlights • HoBi-like pestivirus was administered to pregnant sheep. • Infected animals displayed reproductive disorders. • Persistently infected lambs were born that shed the virus through all body fluids. • Sheep may serve as an alternative model for HoBi-like pestivirus vaccine studies., In order to evaluate sheep as experimental model to test the efficacy of HoBi-like pestivirus vaccines for cattle, 10 sheep at different stages of pregnancy (30 or 50 days) were experimentally infected with the Italian prototype isolate Italy-1/10-1. Irrespective of the stage of pregnancy, virus inoculation resulted in reproductive failures, consisting of abortion, stillbirths or birth of weak or persistently infected (PI) lambs. Aborted fetuses, stillborn and dead lambs displayed extensive histopathological changes, consisting of hemorrhages, congestion and mononuclear infiltration in major organs. Pestiviral antigens were detected by immunohistochemistry in most tissues with remarkable signals in lungs and kidneys. PI lambs were constantly viremic, shed the virus through the nasal secretions and feces and, in all cases but one, did not have detectable HoBi-like pestivirus antibodies before the assumption of colostrum. The single seropositive infected lamb showed low-titer viremia and viral shedding that ceased only several weeks after the 3-month observation period. The study proves that sheep are susceptible to the reproduction failures caused by HoBi-like pestivirus infection and can serve as a suitable model for the evaluation of the fetal protection induced by homologous experimental vaccines.

A calicivirus was detected in neonatal calves with enteritis in Kırklareli, Thrace, Turkey. In the full-length genome, Kırklareli virus was related (48% nucleotide identity) to bovine enteric caliciviruses ( Nebovirus genus). The virus was also detected in a herd in Ankara, Central Anatolia, but not in other Turkish prefectures.

Caliciviruses are important human and animal pathogens. Novel caliciviruses have been identified recently in dogs, raising questions about their pathogenic role and concerns regarding their zoonotic potential. By screening stool samples of young or juvenile dogs using RT-PCR assays, sapoviruses (SaVs) were found in 7/320 (2.2%) samples of animals with acute gastroenteritis while they were not detected in healthy animals (0/119). The sequence of a nearly 3 kb portion at the 3′ end of the genome, encompassing the RNA-dependent RNA polymerase (RdRp), the capsid region (ORF1) and the ORF2 were determined for three strains. A distinctive genetic feature in canine SaVs was a 4-nucleotide (nt) interval between the ORF1 and ORF2. Two strains (Bari/4076/07/ITA and Bari/253/07/ITA) were very closely related in the RdRp and capsid regions to the strain AN210D/09/USA (90.4–93.9% nt), while strain Bari/5020/07/ITA displayed only 71.0–72.0% nt identity to this group of canine SaVs and 76.0% to strain AN196/09/USA. Overall, these findings indicate that the canine SaVs detected in Italy may represent distinct capsid types, although all currently known SaVs segregate into the novel proposed genogroup, tentatively named as GXIII., Highlights • Sapoviruses (SaVs), Caliciviridae family, have been recently discovered in dogs. • Canine SaVs were detected in 2.2% of diarrheic dogs but not in asymptomatic dogs. • The viruses were genetically related to canine SaV prototypes detected in USA.

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.

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. Methods Work involving infectious SARS-CoV was performed in biosafety level 3 facilities. SARS-CoV was grown in monkey VERO cells and human lung epithelial A549 cells, while canine coronavirus (CCoV) was grown in A72 canine cells. Antiviral activity was analysed by determining infective virus titres by TCID50, viral RNA synthesis by Northern blot analysis and real-time RT-PCR, and viral protein synthesis by SDS-PAGE analysis after 35S-methio-nine-labelling. Antiviral efficacy in vivo was determined by evaluating virus titres in CCoV-infected dogs treated orally with 1 mg/kg body weight indomethacin (INDO). Results Unexpectedly, we found that INDO has a potent direct antiviral activity against the coronaviruses SARS-CoV and CCoV. INDO does not affect coronavirus binding or entry into host cells, but acts by blocking viral RNA synthesis at cytoprotective doses. This effect is independent of cyclooxygenase inhibition. INDO's potent antiviral activity (>1,000-fold reduction in virus yield) was confirmed in vivo in CCoV-infected dogs. Conclusions The results identify INDO as a potent inhibitor of coronavirus replication and suggest that, having both anti-inflammatory and antiviral activity, INDO could be beneficial in SARS therapy.

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.

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 × 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. © 2006 Elsevier B.V. All rights reserved.

Coronavirus replication offers several attractive targets for chemotherapy. These include: viral entry (inhibited by chloroquine and peptides); viral RNA (targeted by antisense approaches/RNAi); the main protease 3CLpro (inhibited by peptidic molecules such as HIV-1 protease inhibitors and miscellaneous compounds); the accessory protease(s) PLpro(s) (inhibited by zinc ions); RNA-dependent RNA polymerase (inhibited by aurintricarboxylic acid and antisense approaches); and helicase (inhibited by bananins). Chloroquine and HIV-1 protease inhibitors (with well-known toxicity profiles) should be considered for clinical tests if severe acute respiratory syndrome (SARS) re-emerges; however, there are other attractive compounds. Lessons should be learnt from AIDS research for choosing the best strategies.

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.

Taking into account the close antigenic relationship between bovine herpesvirus 1 (BoHV-1) and caprine herpesvirus 1 (CpHV-1), a live attenuated glycoprotein E (gE) negative BoHV-1 vaccine was assessed in goats with the aim to protect against CpHV-1 infection. Vaccine safety was evaluated by intranasal inoculation of two groups of goats with either a gE-negative BoHV-1 vaccine or a virulent BoHV-1. The length of viral excretion and the peak viral titre were reduced with the gE-negative vaccine. To assess the efficacy, two goats were inoculated intranasally twice 2 weeks apart with a gE-negative BoHV-1 vaccine. Four weeks later, immunised and control goats were challenged with CpHV-1. A 2 log 10 reduction in the peak viral titre was observed and the challenge virus excretion lasted 2 days more in immunised than in control goats. These data indicate the safety and the partial efficacy of a live attenuated gE-negative BoHV-1 vaccine intranasally administrated in goats.

The production of specific antibodies (IgG), interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) was evaluated in dogs in relation to behavioural lateralisation as assessed by paw preference. Left-handed, right-handed and ambidextrous dogs of mixed breed were selected on the basis of their performance in a task consisting of the removal of a piece of adhesive paper from the snout. All dogs were immunised with rabies vaccine. IgG anti-rabies antibody response was evaluated by indirect immunofluorescence (IIF) test. Serum IFN-gamma and IL-10 levels were measured by ELISA in animals showing significant individual left-, right- or no-paw preferences in the behavioural test. The results showed that the direction of behavioural lateralisation influenced the immune response in dogs. The titers of anti-rabies antibodies were lower in left-pawed dogs than in right-pawed and ambidextrous dogs. Similarly, the IFN-gamma serum levels were lower in left-pawed dogs than in right-pawed and ambidextrous dogs. IL-10, on the contrary, seemed to be an immune parameter, which was not affected by lateralisation. These findings suggest that immunomodulation can be correlated with brain laterality in canine species by the regulation of the production of antibodies and some cytokines like IFN-gamma, which are molecules involved in the immune-neurohumoral crosstalk.

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 (from10 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 (HIor = 80) usually considered fully protective for CPV infection, and suggest the need for revision of current vaccination programmes for pups.

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.

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.

Recent identification of unusual canine parvovirus (CPV) mutants in cats and dogs suggests that CPV type 2 (CPV-2), which emerged suddenly in the late 1970s, is undergoing continual genetic and antigenic variations. A peculiarity of parvoviruses is that single-nucleotide substitutions may determine drastic phenotypic changes. The effects of either natural or artificial mutations on CPV phenotypic properties have been largely investigated, and this sets up CPV as an interesting model to study virus evolution. By monitoring the evolution of CPV-2 in Italy, we observed the onset and quick spread of a Glu-426 mutant, antigenically different from the pre-existing variants that were partially displaced within a few years of the initial identification of the new mutant. The identification of CPV-2 variants raises several questions concerning their impact on the efficacy of the current CPV-2 vaccines, based on the original CPV-2 strain that no longer exists in the field.

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.

During an epidemiological survey encompassing several porcine herds in Saragoza, Spain, the VP7 and VP4 of a rotavirus-positive sample, 34461-4, could not be predicted by using multiple sets of G- and P-type-specific primers. Sequence analysis of the VP7 gene revealed a low amino acid (aa) identity with those of well-established G serotypes, ranging between 58.33% and 88.88%, with the highest identity being to human G2 rotaviruses. Analysis of the VP4 gene revealed a P[23] VP4 specificity, as its VP8* aa sequence was 95.9% identical to that of the P14[23],G5 porcine strain A34, while analysis of the VP6 indicated a genogroup I, that is predictive of subgroup I specificity. Analysis of the 10th and 11th RNA segments revealed close identity to strains of porcine and human origin, respectively. The relatively low overall aa sequence conservation (

Beta2 (β2) toxin–producing Clostridium perfringens type A strains were found to be associated with necrotic and hemorrhagic intestinal lesions in 2 Asiatic black bears ( Selenarctos thibetanus) that died suddenly. Ten isolates were obtained from the liver, lungs, heart, and small and large intestine of the animals and were examined by multiplex polymerase chain reaction for the genes encoding the 4 lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types as well as for the genes encoding enterotoxin and the novel β2-toxin for subclassification. In addition, the cpb2 sequence of the 10 isolates was different from the published sequence of cpb2 of pig type C isolate CWC245, whereas it was highly similar to the cpb2 sequence of the C. perfringens type A strain 13. This finding suggests the existence of 2 cpb2 subtypes. This is the first report of enterotoxemia associated with the presence of C. perfringens producing β2-toxin in the tissues and intestinal content of Asiatic black bears.

A clinical outbreak of bovine piroplasmosis was reported in Italy. The etiological agent was characterized as Babesia occultans , a parasite regarded as apathogenic and never detected before in continental Europe. This report paves the way for further studies to assess the occurrence of this tick-transmitted protozoan in other European regions.

Hobi-like pestivirus, a new tentative species within genus Pestivirus, was firstly detected in foetal bovine serum batches and later associated to respiratory distress and reproductive failures in cattle. In the present study, the cross-antibody response between bovine viral diarrhoea virus 1 (BVDV-1) and the emerging pestivirus was evaluated in the sheep model. Ten sheep were immunised against BVDV-1 or Hobi-like pestivirus using inactivated preparations and the induced antibody responses were evaluated against the homologous and heterologous viruses. The results showed that heterologous antibody titres were significantly lower than the homologous ones, thus suggesting the need to develop specific vaccines against the emerging pestiviral species.

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.

Aims: To provide information on epidemiology and isolation of Salmonella strains from reptiles. Methods and Results: Ninety-one samples collected from reptiles of the zoo of Rome or belonging to private owners were analysed using a standard protocol for isolation of Salmonella from food. Salmonella strains were tested for susceptibility to 15 antimicrobics by a disc-agar diffusion method. Forty-six samples (50·5%) were positive for Salmonella. Of the 22 strains serotyped, 17 belonged to Salmonella enterica subsp. I, four to the subsp. IIIa and one strain resulted untypeable. Rappaport–Vassiliadis broth (RVB) allowed to recover more Salmonella strains when bacterial growth in buffered peptone water (BPW) was scarce, while selenite cystine broth (SCB) was more efficient, whereas growth in BPW was abundant. The maximum isolation score was obtained by plating onto xylose lysine desoxycholate agar (XLD). The strains exhibited resistance at high percentages to colistin sulphate (58·7%), sulphamethoxazole (55·5%), streptomycin (32·6%), tetracycline (19·6%), ampicillin (17·4%) and nalidixic acid (13·1%). Conclusions: A high prevalence of Salmonella in reptiles was observed. For isolation, the choice of the enrichment broth depending on the degree of growth in BPW followed by plating onto XLD may be suggested. Significance and Impact of the Study: This paper provides epidemiological data on the prevalence of Salmonella and laboratory protocols useful for isolation of Salmonella from faeces of reptiles.

The immunogenicity of an inactivated oil-emulsion vaccine against canine distemper virus was evaluated in nine captive African wild dogs (Lycaon pictus). Antibody levels were determined by neutralization test in Vero cells. No significant local or systemic adverse reactions were observed in the animals. Virus neutralizing antibody levels >1:20 were detected, especially in animals that were vaccinated twice. The use of oil adjuvants is suggested as a good way to enhance the immune response to inactivated canine distemper vaccine.

By antigenic and genetic characterization of canine parvovirus type 2 (CPV-2) strains collected in 2001 and 2002 in Italy, it was possible to observe the spread of viruses with an unusual mutation, Glu-426, affecting a major antigenic epitope of CPV-2. Out of 67 strains analyzed, 49 (73.13%) were characterized as CPV-2a, 6 (8.95%) were characterized as CPV-2b, and 12 (17.91%) were characterized as the Glu-426 mutant.

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.

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].

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.

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.