TNF-alpha levels in tears: a novel biomarker to assess the degree of diabetic retinopathy.

We assess the level of tumour necrosis factor alpha (TNF-alpha) in tear fluids and other serum parameters associated with diabetes in different degrees of diabetic retinopathy. We have performed a prospective, nonrandomized, observational study. Study population consisted of 16 healthy subjects (controls) and 32 type 2 diabetic patients: 16 affected by proliferative diabetic retinopathy (PDR) and 16 with nonproliferative retinopathy (NDPR, background/preproliferative). Body mass index, urinary albumin, blood glucose, HbA1c, and tear levels of TNF-alpha were measured in all subjects. The value of glycaemia, microalbuminurea, and Body mass index in diabetic retinopathy groups were higher than those in control group (P < 0.05). Glycemia in NPDR: 6.6 mmol/L (range: 5.8-6.3); in PDR: 6.7 mmol/L (range: 6.1-7.2); in control: 5.7 mmol/L (range: 4.9-6.1); microalbuminurea in NPDR: 10.6 mg/L (range: 5.6-20); in PDR: 25.2 mg/L (range: 17-40); in control: 5.3 mg/L (range: 2.6-10); Body mass index in NPDR: 26 Kg/m(2) (range: 20.3-40); in PDR: 28 Kg/m(2) (range 20.3-52); in control: 21 Kg/m(2) (range 19-26). The TNF-alpha concentrations in tears increase with the severity of pathology and were lower in control group than in diabetic subjects. In the end, the level of TNF-alpha is highly correlated with severity of diabetic retinopathy and with nephropathy. Tear fluid collection may be a useful noninvasive method for the detection of proliferative diabetic retinopathy.

The evolutionary dynamics of transposable elements in eukaryote genomes.

Transposable elements (TEs) are ubiquitous components of eukaryotic genomes. They have considerably affected their size, structure and function. The sequencing of a multitude of eukaryote genomes has revealed some striking differences in the abundance and diversity of TEs among eukaryotes. Protists, plants, insects and vertebrates contain species with large numbers of TEs and species with small numbers, as well as species with diverse repertoires of TEs and species with a limited diversity of TEs. There is no apparent relationship between the complexity of organisms and their TE profile. The profile of TE diversity and abundance results from the interaction between the rate of transposition, the intensity of selection against new inserts, the demographic history of populations and the rate of DNA loss. Recent population genetics studies suggest that selection against new insertions, mostly caused by the ability of TEs to mediate ectopic recombination events, is limiting the fixation of TEs, but that reduction in effective population size, caused by population bottlenecks or inbreeding, significantly reduces the efficacy of selection. These results emphasize the importance of drift in shaping genomic architecture.

Safe use of vaccines and vaccine compliance with food safety requirements.

Advanced technologies and regulatory regimes have contributed to the availability of veterinary vaccines that have high quality and favourable safety profiles in terms of potential risks posed to the target animals, the persons who come into contact with the vaccine, the consumers of food derived from vaccinated animals and the environment. The authorisation process requires that a range of safety studies are provided to evaluate the products. The design and production of vaccines, and their safe use, are primarily assessed by using data gathered from extensive pre-marketing studies performed on target animals and specific quality tests. The current post-marketing safeguards include good manufacturing practices, batch safety testing, inspections and pharmacovigilance. In addition to hazard identification, a full benefit/risk evaluation needs to be undertaken. The outcome of that evaluation will determine options for risk management and affect regulatory decisions on the safety of the vaccine; options might, for example, include special warnings on package inserts and labels.

Validation of rt-PCR assays for molecular characterization of porcine teschoviruses and enteroviruses.

Porcine enteroviruses (PEVs) and teschoviruses (PTVs) are described as causative agents of neurological disorders, fertility disorders and dermal lesions of swine. Difficulties in the serological detection of these viruses may lead to a significant underestimation of infections with clinical symptoms. With the recent availability of genome sequence data for all the serotypes, molecular diagnosis is a possibility. The present study describes a new approach to molecular 'serotyping' of PTVs and PEV-B viruses, involving the amplification and sequencing of a genomic fragment of the VP1 coding region. A molecular characterization of Italian entero-teschovirus isolates was performed using a set of previously published and newly designed polymerase chain reaction primers. A total of 33 porcine isolates and 10 reference strains were analysed. Porcine enterovirus-B samples were first diagnosed as positive for enterovirus by amplification of the 5'-non-translated region. Samples were then typed by amplification and sequencing of a portion of the VP1 coding region. Porcine enterovirus-A and PTVs were detected by a published assay in the 5'-NC region that allows them to be differentiated according to the size of amplification product, using the same set of primers. For serotype characterization of PTV, we evaluated four different regions: the N terminus of the capsid protein VP2, the region encoding for RNA-dependent RNA polymerase, and the capsid VP1 and VP4 regions. The newly designed primers in the VP1 region was proved to be broad in range and suitable for serotype assessment and therefore constitute a useful diagnostic tool for molecular diagnosis of porcine teschovirus/enterovirus strains and for the study of molecular epidemiology and evolution of these viruses.

Current concepts and future approaches to the development of autologous/autogenous vaccines for veterinary use.

Current classification of autologous/autogenous (A/A) vaccines is commonly based on the concept of strain/antigen specificity associated with targeted treatment of a restricted number of animals. However, fulfilling these two conditions is not sufficient for immune-veterinary immunebiologicals to be excluded from the provisions of Directive 2001/82/EC. Indeed, non-inactivated A/A vaccines are not automatically considered out of the scope of the community code relating to veterinary medicinal products, in particular to immune-biologicals. As a major consequence of the "regulatory" exclusion from the requirements of EU rules, A/A vaccines can be usually manufactured and distributed without having obtained a marketing authorization by the competent authority of a Member State. Furthermore, strain specificity enables veterinarians to consider the use of these vaccines in quite a large variety of epidemiological circumstances where no "conventional" vaccines are yet available or are considered efficacious. In addition, in contrast to "conventional" vaccines, which are considered exclusively as a preventive tool against infectious diseases, A/A vaccines can also be used to treat "continuing" infections. Although the limited scientific value of these products and the poor investigations of the effector mechanisms involved are widely recognized, their use is still claimed in conditions where disorders in the immune system are suspected. Today, a more appropriate definition of A/A vaccines is one that takes into account their historical tradition and practical use, such as stable- or herd-specific vaccines, custom ("..ized") vaccines, therapeutic vaccines, pharmavaccines, vaccines used for biological therapy, etc. Although acknowledging the "regulatory autonomy" of A/A vaccines versus "conventional" vaccines, here it will be presented as an overview of the necessary points to consider, to guarantee an acceptable standard in the development and control of this particular category of veterinary immune-biologicals.

Molecular characterization of low pathogenicity H7N3 avian influenza viruses isolated in Italy.

The complete coding regions of the surface glycoproteins, nucleoprotein (NP), polymerase 2 (PB2), and matrix (M) of A/turkey/214845/02 and A/turkey/220158/99 (H7N3) low pathogenicity avian influenza (LPAI) viruses isolated in October 2002 in Italy were amplified and sequenced to determine the epidemiologic relationships with an A/turkey/Italy/4603/99 (H7N1/4603/99) LPAI virus isolated during the 1999-2001 epizootic in Italy. The hemagglutinin (HA) of H7N3 viruses showed 97.8% nucleotide similarity with A/turkey/Italy/4603/99 (H7N1), and NP, M, and PB2 gene similarities were 93.6%, 98.2%, and 96.2%, respectively. Phylogenetic analyses of HA, PB2, and M genes showed that H7N3 and H7N1 viruses were closely related. Sequence analysis revealed a 23 amino acid deletion in the stalk of the neuraminidase of H7N3 viruses and a unique deletion of amino acid glycine in position 17 in the NP gene of H7N1 virus.

Influenza surveillance in birds in Italian wetlands (1992-1998): is there a host restricted circulation of influenza viruses in sympatric ducks and coots?

We report the results of a 6-year serological and virological monitoring performed in ducks and coots in Italy, in order to assess the degree of influenza A virus circulation in these birds during wintering. A total of 1039 sera collected from 1992 to 1998 was screened by a double antibody sandwich blocking ELISA (NP-ELISA): seroprevalence of antibodies to influenza A viruses was significantly higher in ducks compared to coots (52.2% vs. 7.1%, respectively). The hemagglutination-inhibition (HI) assay, performed on NP-ELISA positive sera, showed that 16.9% of these duck sera and 33.3% of these coot sera had antibodies to at least one influenza virus HA subtype: ducks showed HI antibodies against most of the HA subtypes, except for the H3, H4, H7, and H12; coots were seropositive to the H3 and H10 subtypes, only. From 1993 to 1998, 22 virus strains were obtained from 802 cloacal swabs, with an overall virus isolation frequency of 2.7%. Viruses belonging to the H1N1 subtype were by far the most commonly circulating strains (18/22) and were isolated mainly from ducks (17/18). The remaining viruses were representative of the H10N8, H5N2 and H3N8 subtypes. Our data indicate some differences between influenza A virus circulation in sympatric ducks and coots and a significant antigenic diversity between some reference strains and viruses recently isolated in Italy.

Experimental infection of calves with bovine viral diarrhoea virus type-2 (BVDV-2) isolated from a contaminated vaccine.

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100%, homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.

Genetic heterogeneity of bovine viral diarrhoea virus in Italy.

The genetic characteristics, of 38 field isolates of bovine viral diarrhoea virus (BVDV) collected in 1999 from sick or healthy and persistently infected cattle of dairy farms situated in northern Italy, were investigated. A partial 5'-untranslated region (5'-UTR) sequence of each isolate was determined and a phylogenetic analysis was performed. All the isolates were classified as belonging to the BVDV-1 genotype and could be assigned to different BVDV-1 groups, namely BVDV-1b (n = 20), BVDV-1d (n = 6) and BVDV-1e (n = 10). Two remaining isolates could be classified as BVDV-1f and BVDV-1h, respectively. These results provided evidence for genetic heterogeneity of BVDV in Italy, and contribute to a better knowledge of the circulation of BVDV strains, and to their classification.

Standardization of an inactivated H17N1 avian influenza vaccine and efficacy against A/Chicken/Italy/13474/99 high-pathogenicity virus infection.

The minimum requirements for assessing the immunogenicity of an experimental avian influenza (AI) vaccine prepared from inactivated A/Turkey/Italy/2676/99 (H7N1) low-pathogenicity (LP) AI (LPAI) virus were determined in chickens of different ages. A correlation between the amount of hemagglutinin (HA) per dose of vaccine and the protection against clinical signs of disease and infection by A/Chicken/Italy/13474/99 highly pathogenic (HP) AI (HPAI) virus was established. Depending on the vaccination schedule, one or two administrations of 0.5 microg of hemagglutinin protected chickens against clinical signs and death and completely prevented virus shedding from birds challenged at different times after vaccination.

ELISA test for the detection of influenza H7 antibodies in avian sera.

Using a monoclonal antibody (MAb) specific for the H7 influenza surface glycoproteins, a serological enzyme-linked immunosorbent assay (ELISA) test has been developed. This MAb was made using the low-pathogenicity (LP) avian influenza (AI) strain (BS2676/99) isolated in Italy during a recent outbreak. The test is able to detect H7 antibodies in avian sera. The H7 ELISA has a 99% concordance of results with the classical hemagglutination inhibition (HI) test.

Recent developments in avian influenza research: epidemiology and immunoprophylaxis.

Influenza A viruses have been isolated from humans, from several other mammalian species and a wide variety of avian species, among which, wild aquatic birds represent the natural hosts of influenza viruses. The majority of the possible combinations of the 15 haemagglutinin (HA) and nine neuraminidase (NA) subtypes recognized have been identified in isolates from domestic and wild birds. Infection of birds can cause a wide range of clinical signs, which may vary according to the host, the virus strain, the host's immune status, the presence of any secondary exacerbating microorganisms and environmental factors. Most infections are inapparent, especially in waterfowl and other wild birds. In contrast, infections caused by viruses of H5 and H7 subtypes can be responsible for devastating epidemics in poultry. Despite the warnings to the poultry industry about these viruses, in 1997 an avian H5N1 influenza virus was directly transmitted from birds to humans in Hong Kong and resulted in 18 confirmed infections, thus strengthening the pandemic threat posed by avian influenza (AI). Indeed, reassortant viruses, harbouring a combination of avian and human viral genomes, have been responsible for major pandemics of human influenza. These considerations warrant the need to continue and broaden efforts in the surveillance of AI. Control programmes have varied from no intervention, as in the case of the occurrence of low pathogenic (LP) AI (LPAI) viruses, to extreme, expensive total quarantine-slaughter programmes carried out to eradicate highly pathogenic (HP) AI (HPAI) viruses. The adoption of a vaccination policy, targeted either to control or to prevent infection in poultry, is generally banned or discouraged. Nevertheless, the need to boost eradication efforts in order to limit further spread of infection and avoid heavy economic losses, and advances in modern vaccine technologies, have prompted a re-evaluation of the potential use of vaccination in poultry as an additional tool in comprehensive disease control strategies. This review presents a synthesis of the most recent research on AI that has contributed to a better understanding of the ecology of the virus and to the development of safe and efficacious vaccines for poultry.

Genotyping of bovine viral diarrhoea viruses isolated from cattle in northern Italy.

Following the first official report of a clinically severe outbreak of bovine viral diarrhoea disease occurring in a farm in northern Italy, which had originated from the use of a live vaccine contaminated with a strain of BVD genotype II virus, a retrospective study on the prevalence of BVDV genotypes in Italy became highly relevant. For this purpose, the genotype of 78 BVDV-positive specimens, obtained in 1998-1999 from dairy cattle in an area near to where the outbreak occurred, was characterized by PCR technology. Two sets of primers, spanning the 5' UTR of BVDV genome, were used sequentially in a first round of RT-PCR, performed on viral RNA extracted directly from 15 clinical samples and 63 BVDV-infected cell-culture fluids; a second PCR assay followed to selectively amplify only BVDV genotype II. All the viruses under study were characterized as BVDV genotype I. As well as contributing to a better understanding of the prevalence of BVDV genotypes in the field, the results of the present study illustrate the possibility that novel BVDV strains can emerge in susceptible animals through the use of contaminated immunobiological products for bovine use.

Determination of bovine rotavirus G and P serotypes in italy by PCR.

Determination of the G and P serotypes of group A bovine rotaviruses from 149 samples of feces or intestinal contents collected from calves showing clinical signs of neonatal diarrhea was performed by a nested reverse transcription-PCR typing assay. The G6 serotype was the most prevalent, accounting for viruses in 55.7% of the samples; viruses of the G10 and G8 serotypes were found in 34.9 and 4.7% of the samples, respectively. The virus in one sample (0.7%) was not classified due to concomitant infection with G6 and G8 strains, whereas viruses in six samples (4.0%) could not be characterized with any of the three G serotype-specific primers selected for the present study. When examined for their P-serotype specificities, viruses in 55 and 42.3% of the samples were characterized as P[11] and P[5], respectively, no P[1] serotype was identified, and viruses in 2.7% of the samples could not be classified due to multiple reactivity with both P[5]- and P[11]-specific primers. Various combinations of G and P serotypes were observed, the most frequent being G6,P[5] (38.3%), G10,P[11] (31.5%), and G6,P[11] (15.4%). The results of the present study, while contributing to a better understanding of the epidemiology of bovine rotaviruses in Italy, address the relevance of serotype specificity with regard to the constancy of the quality of bovine rotavirus vaccines under different field conditions.

Comparative evaluation of in vitro and in vivo assays for the detection of avian infectious bronchitis virus as a contaminant of live poultry vaccines.

A reverse transcriptase polymerase chain reaction (RT-PCR( assay specific for identifying avian infectious bronchitis virus (IBV) in poultry vaccines, and the serological response to IBV induced by the inoculation of chicks with a Newcastle disease vaccine spiked with the Massachusetts strain of IBV, were compared for their ability to detect IBV as a contaminant of avian vaccines. The sensitivity of the IBV-RT-PCR assay provided results which were at least equivalent to the biological effect produced by the inoculation of chicks, allowing this assay to be considered a valid alternative to animal testing in the quality control of avian immunologicals. This procedure can easily be adapted to detect a number of contaminants for which the in vivo test still represents the only available method of detection.

Rapid diagnosis of avian infectious bronchitis virus by the polymerase chain reaction.

A simple, sensitive and specific polymerase chain reaction (PCR) procedure was developed in order to detect infectious bronchitis virus (IBV) directly in tissue samples. Viral RNA was extracted from allantoic fluids and cell cultures infected experimentally with different strains of IBV and from tissues of naturally infected birds. Viral RNA was then amplified and identified by a nested RT-PCR assay using two sets of primers flanking a well-conserved region of the nucleocapsid gene. The selected IBV nucleocapsid sequence was detected successfully by simple direct electrophoresis of amplified material.

Serological responses in calves to vaccines against bovine respiratory syncytial, infectious bovine rhinotracheitis, bovine viral diarrhoea and parainfluenza-3 viruses.

The Istituto Superiore di Sanità (ISS), the National Veterinary Services Laboratory in Italy, is in charge of assessing the quality, safety and efficacy of veterinary vaccines before and after licensing. To evaluate the relative potency of several vaccines against bovine respiratory syncytial virus (BRSV), infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhoea virus (BVDV) and parainfluenza-3 virus (PI3V), the serological responses in vaccinated calves were studied. Vaccination with any of the vaccines under study induced specific antibody titres against the different viral antigens. The differences of the mean antibody titres within and among the test group vaccines were statistically significant. The results confirm and support those obtained by other authors in similar studies, suggesting that serological responses in vaccinated calves can be used as a helpful means of assessing the relative potency of vaccines against viral respiratory diseases of cattle. The criteria allowing such an evaluation are discussed.