Identification and characterisation of Gamma-herpesviruses in zoo artiodactyla.

BACKGROUND: Viruses within the γ-herpesviruses subfamily include the causative agents of Malignant Catarrhal Fever (MCF) in several species of the order Artiodactyla. MCF is a usually fatal lymphoproliferative disease affecting non-adapted host species. In adapted host species these viruses become latent and recrudesce and transmit during times of stress or immunosuppression. The undetected presence of MCF-causing viruses (MCFVs) is a risk to non-adapted hosts, especially within non-sympatric zoological collections. This study investigated the presence of MCFVs in six different zoological collections in the UK, to evaluate the presence of subclinical/latent MCFVs in carrier animals. METHODS: One-hundred and thirty eight samples belonging to 54 different species of Artiodactyla were tested by Consensus Pan-herpes PCR. The positive samples were sequenced and subjected to phylogenetic analyses to understand their own evolutionary relationships and those with their hosts. RESULTS: Twenty-five samples from 18 different species tested positive. All viruses but one clustered in the γ-herpesvirus family and within the Macavirus as well as the non-Macavirus groups (caprinae and alcelaphinae/hippotraginae clusters, respectively). A strong association between virus and host species was evident in the Macavirus group and clustering within the caprinae group indicated potential pathogenicity. CONCLUSION: This study shows the presence of pathogenic and non-pathogenic MCFVs, as well as other γ-herpesviruses, in Artiodactyla species of conservation importance and allowed the identification of new herpesviruses in some non-adapted species.

Partial Replacement of Synthetic Vitamin E by Polyphenols in Post-Weaning Piglets.

Vitamin E is an essential nutrient usually recommended in post-weaning piglets, when a decline in the serum vitamin E concentration is observed. Selected polyphenols have the potential to partially replace vitamin E in animal feed. The aim of this study was to investigate the effect of the dietary inclusion of some commercial polyphenol products (PPs) on the growth performance, antioxidant status and immunity of post-weaning piglets. A total of 300 piglets (BW 7.18 kg ± 1.18) were randomly assigned to six dietary groups: CON- (40 mg/kg vitamin E); CON+(175.8 mg/kg vitamin E); and PP1, PP2, PP3 and PP4, in which 50% vitamin E of CON+ was replaced with PP with equivalent vitamin E activity. The PP1 group exhibited lower performance (p < 0.05) than the other dietary groups, but a similar performance to that commonly registered in pig farms. Dietary polyphenols did not influence the IgG concentration or the IL-6, IL-10, IFN-γ and TNF-α cytokine concentrations. A lower IL-8 level was found in the PP4 group than in the other groups. The diets that affected the vitamin A content showed the highest value (p < 0.05) in the PP1 group, and a trend was noted for vitamin E with a higher content in PP4 and CON+. The polyphenols-enriched diets, especially the PP3 diet, maintained an antioxidant capacity (whole blood KRL) similar to the CON+ diet. In conclusion, the replacement of vitamin E with all PPs enables partial vitamin E substitution in post-weaning piglets.

Effect of Aging on the Immune Response to Core Vaccines in Senior and Geriatric Dogs.

Elderly dogs are steadily increasing worldwide as well as veterinarians' and owners' interest in their health and wellness. Aging is not a disease, but a combination of changes negatively affecting the organism in general and the immune system in particular, resulting in a decline in protection over time. The aim of this study was to measure the specific serum antibody titers against the main dangerous and widespread viral diseases preventable by core vaccinations in senior and geriatric dogs using the in-practice test VacciCheck. A cohort of three hundred fifty elderly dogs was analyzed for Protective Antibody Titers (PATs) against CPV-2, CDV and CAdV-1. The age ranged from 5 to 19 years, with two hundred fifty-eight seniors (73.7%) and ninety-two geriatrics (26.3%), and 97.4% of them were vaccinated at least once in their lives. More than half of the entire study population (52.9%) had PATs simultaneously for all three diseases, with 80.5% seniors and 19.5% geriatrics. Specific PATs were found in 88.6% of aging dogs for CPV-2, 82.3% for CadV-1 and 66.0% for CDV, demonstrating that unprotected aging dogs represent a minority. Unexpectedly, the larger elderly dogs resulted as more protected than smaller ones for CPV-2. Protection then decreases over time, with geriatric dogs less protected than senior ones. Veterinary practitioners should therefore always consider whether to maintain core vaccinations in aging dogs as in adults on a three-year basis or opt instead for closer boosters (every 1 or 2 years). PATs for core vaccines could then represent a good biomarker of protection and their titration could become a standard of care, especially in such a sensitive period of the dogs' life.

Can Chemotherapy Negatively Affect the Specific Antibody Response toward Core Vaccines in Canine Cancer Patients?

The life expectancy of our pets has been getting longer in recent years due to new therapeutic opportunities, better nutrition, and better diagnostic approaches. This positive effect, however, has been accompanied by a concomitant increase in neoplasms, particularly in canine patients. Therefore, veterinarians inevitably face new issues related to these diseases, poorly or never investigated in the past, such as the possible side effects resulting from chemotherapy. The aim of this study was to investigate whether and how chemotherapy influences the antibody response against CPV-2, CDV, and CAdV-1 in dogs vaccinated before starting chemotherapy. Twenty-one canine patients with different types of malignancies were sampled before, during, and after different chemotherapy protocols to determine their actual levels of seroprotection against CPV-2, CDV, and CadV-1 by using the in-practice test VacciCheck. Differences related to sex, breed size, type of tumor, and chemotherapy protocol were evaluated. No statistically significant changes in antibody protection emerged for any of the chemotherapy protocol used, suggesting that, contrary to expectation, chemotherapy does not have a marked immunosuppressive effect on the post-vaccine antibody response. These results, although preliminary, may be useful in improving the clinical approach to the canine cancer patient, helping veterinarians fully manage their patients, and enabling owners to feel more confident about their pets' quality of life.

Tobacco Seed-Based Oral Vaccination against Verocytotoxic O138 Escherichia coli as Alternative Approach to Antibiotics in Weaned Piglets.

Post-weaning diarrhoea and enterotoxaemia caused by Escherichia coli are serious threats in the pig (Sus scrofa domesticus) livestock industry and are responsible for economic losses related to mortality, morbidity and stunted growth. The aim of this study was to evaluate the effect of an engineered tobacco seeds-based edible vaccine in O138 Escherichia coli-challenged piglets throughout a multidisciplinary approach. Thirty-six weaned piglets were enrolled and randomly divided into two experimental groups, a control (C; n = 18) group and a tobacco edible vaccination group (T, n = 18), for 29 days of trial. At days 0, 1, 2, 5 and 14, piglets of the T group were fed with 10 g of the engineered tobacco seeds line expressing F18 and VT2eB antigens, while the C group received wild-type tobacco seeds. After 20 days, 6 piglets/group were orally challenged with the Escherichia coli O138 strain (creating four subgroups: UC = unchallenged control, CC = challenged control, UT = unchallenged tobacco, CT = challenged tobacco) and fed with a high protein diet for 3 consecutive days. Zootechnical, clinical, microbiological, histological and immunological parameters were assayed and registered during the 9 days of post-challenge follow up. At 29 days post-challenge, the CT group displayed a lower average of the sum of clinical scores compared to the CC group (p < 0.05), while the CC group showed a higher average sum of the faecal score (diarrhoea) (p < 0.05) than the CT group. A decreased number of days of shedding of the pathogenic strain was observed in the CT compared to the CC group (p < 0.05). Specific anti-F18 IgA molecules were significantly higher in the CT group compared to the CC group's faecal samples during the post-challenge period (p < 0.01). In conclusion, edible vaccination with engineered tobacco seeds showed a protective effect on clinical symptoms and diarrhoea incidence during the post-challenge period, characterized by a limited time of pathogenic strain shedding in faeces.

Prevalence of Serum Antibody Titers against Core Vaccine Antigens in Italian Dogs.

Canine vaccination is the main tool for preventing dangerous and widespread diseases. The strongly recommended (core) dog vaccines are against Canine Parvovirus type 2 (CPV-2), Canine Distemper Virus (CDV), and Canine Adenovirus (CAdV-1), but vaccination protocols should be tailored to dog lifestyles. Vaccination guidelines suggest vaccinating adult dogs no more frequently than every 3 years using modified live (attenuated) vaccines (MLV), thus obtaining a long-lasting (sometimes throughout life) specific protection in many but not all animals. The aim of this study was to determine the actual levels of seroprotection against CPV-2, CDV and CAdV-1 in a cohort of Italian dogs by using the in-practice test VacciCheck. A total of 1,027 dogs (951 vaccinated and 76 unvaccinated) were analyzed for Protective Antibody Titers (PATs) against CPV-2, CDV, and CAdV-1. Differences related to sex, age, breed size, health status, and time elapsed since last vaccination were evaluated. Half of the entire canine cohort (50.6%) had PATs for all three viruses (68.5% considering only vaccinated dogs). In particular, 90.8% of dogs were protected against CPV-2, 68.6% against CDV, and 79.8% against CAdV-1. Most dogs remained protected for 3 years after vaccination or longer. Revaccination on a 3-year basis can then be recommended for core MLV vaccines without altering individual's seroprotection or even herd immunity.

Molecular Tools to Identify and Characterize Malignant Catarrhal Fever Viruses (MCFV) of Ruminants and Captive Artiodactyla.

The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested.

In vitro replication activity of bovine viral diarrhea virus in an epithelial cell line and in bovine peripheral blood mononuclear cells.

The present study focused on the in vitro infection of Madin-Darby bovine kidney (MDBK) cells and bovine peripheral blood mononuclear cells (PBMCs) from naÏve animals with non-cytopathic (ncp, BVDV-1b NY-1) and cytopathic (cp, BVDV-1a NADL) strains. Infections with 0.1 and 1 multiplicity of infections (MOI) and incubation times of 18 and 36 hr were compared. Twelve BVDV naÏve heifers were enrolled to collect PBMCs. The viral loads in MDBK cells and in PBMCs after in vitro infections were measured by real-time polymerase chain reaction (PCR) assays. The highest viral loads were measured at 1 MOI and 36 hr post infection in both cell systems and the lowest at 0.1 MOI and 18 hr with the exception of the cp strain NADL in PBMCs, for which the highest viral load was observed at 0.1 MOI and 36 hr. Viral load mean values were higher for the cp strain than the ncp strain irrespective of the extent of the infection period and MOI. The models of infection studied uncovered different replication activities respectively according to the biotype of virus, the cell substrate and the duration of infection. Replication tends to be higher in PBMCs, particularly at low MOIs and for the ncp strain.

Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats.

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis-encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.

Expression of c-kit proto-oncogene in canine mastocytoma: a kinetic study using real-time polymerase chain reaction.

KIT receptor, the c-kit gene product, is thought to play a major role in canine mastocytoma, one of the most common neoplastic diseases in dogs. In the present study, the expression of c-kit proto-oncogene in blood and in tumor biopsies from 41 dogs with histologically confirmed mastocytoma at different grades of cellular differentiation and 5 negative control dogs was investigated using real-time (quantitative) reverse transcription-polymerase chain reaction (RRT-PCR). The animals were followed up for over 1 year after surgery in order to characterize the kinetics of c-kit expression in blood. Transcript mRNAs extracted from blood at different time points after surgery and from tumor tissue surgically removed from each dog were used in a quantitative RRT-PCR assay targeting the extracellular coding region of the c-kit gene. Tissues constitutively expressing c-kit (brain and spleen) were used as positive controls. Levels of expression of c-kit were higher in tumor biopsies than in blood; the blood level decreased in the patients between 1 and 3 months after surgery. No KIT expression was detected in blood from the 5 dogs not affected by mastocytoma (negative controls). The RRT-PCR appears to be a suitable method for sensitive and quantitative detection of c-kit gene expression in canine blood and neoplastic tissues. Although c-kit expression levels measured by RRT-PCR do not correlate with prognosis, they confirm that surgery remains the main treatment to reduce circulating mastocytes and that circulating mast cells can be detected even in benign highly differentiated forms of mastocytoma such as grade I.

A study of mutations in the c-kit gene of 32 dogs with mastocytoma.

Mutations in the intracellular juxtamembrane domain of the c-kit gene in 32 dogs with different grades of histologically confirmed mastocytoma were studied. Transcript RNAs extracted from neoplastic tissue surgically collected from dogs of different breeds and from a negative control were reverse transcribed into complementary DNA and amplified by polymerase chain reaction. The region corresponding to the c-kit juxtamembrane domain was sequenced and compared with GenBank sequences. Two different types of mutations were identified within exon 11: a previously underscribed single-nucleotide substitution and a 6-bp deletion. The c-kit juxtamembrane domain sequences of all dogs were grouped in 3 clusters. No mutations were detected in tissues constitutively expressing c-kit (cerebellum and spleen), obtained from dogs not affected by mastocytoma (controls). All the substitutions were found in dogs bearing grade I or II mast cell tumors; the deletion was detected in 1 dog with grade II mastocytoma.