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.

Shifts in immune responses of an invasive alien species: A test of the evolution of increased competitive ability hypothesis using American Eastern gray squirrels in Italy.

Based on the Evolution of Increased Competitive Ability (EICA) hypothesis, a reduced investment in immunity, consequent to parasite loss, could partly explain the success of invasive alien species. We investigated variation in parasite load and immune responses of alien Eastern gray squirrels (Sciurus carolinensis) along the invasion wave of an expanding population. We first verified by fecal analyses that 1) parasite abundance decreased moving from the core towards the invasion front. Next, we used multiple measures of immunity to investigate whether, in response to the lower parasite pressure, individuals at the invasion front 2) dampened their costly inflammatory response, and 3) increased their investment in less expensive acquired immunity. We first explored variation in hematological variables related either to the inflammatory or the acquired response. On a subset of individuals, we carried out ex vivo cell cultures to analyse the basal expression of MHC class II genes and the expression of TNF-α genes in response to an immune challenge. Platelet counts and TNF-α expression suggested higher inflammation in individuals living at the invasion core, whereas parameters associated with an acquired response (lymphocyte counts and MHC II expression by spleen cells), conversely, were higher in squirrels at the front. Overall, our results suggest a shift between different immune strategies along the invasion wave, supporting a reduced investment in costly inflammatory responses and an increased investment in acquired immunity in individuals at the expanding edge of the range, which are subjected to high selective pressures for dispersal and reproduction.

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.

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.

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.

IL-1R8 Downregulation and Concomitant TLR7 and TLR9 Upregulation Are Related to the Pathogenesis of Canine Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy in humans and dogs. Several studies disclosed some similarities between the two species, including the constitutive activation of the NF-κB pathway as a fundamental underlying pathogenetic mechanism. In humans, the downregulation of IL-1R8 is implicated in DLBCL development, but its role in dogs has not been explored so far. To gain insight into the pathogenesis of this tumor in dogs, we evaluated the mRNA and protein expression of IL-1R8 in 12 control lymph nodes obtained from dogs not bearing tumors and from 50 dogs with DLBCL. Moreover, we analyzed through qRT-PCR the expression of TLR7, TLR9, MYC, and p52 genes that are known to be involved in the IL-1R8 regulatory network. IL-1R8 and p52 were downregulated in DLBCLs compared to control lymph nodes (p < 0.001), while a higher expression of TLR7, TLR9, and MYC was observed in DLBCLs (p < 0.01). Immunohistochemistry confirmed the gene expression results, revealing a significantly lower IL-1R8 staining score in DLBCLs compared to control lymph nodes (p < 0.0001). Taken together, these results suggest that IL-1R8 downregulation may represent one of the mechanisms driving DLBCL pathogenesis in dogs, mainly through the dysregulation of the Toll-like/interleukin receptors signaling cascade and the aberrant activation of the classical NF-κB pathway.

Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period.

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-ß (IL-1ß), Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors.

Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells.

Mitogens are diverse compounds of plant and microbial origin, widely employed to test immunocompetence in animals. The blastogenic response of bovine Peripheral Blood Mononuclear Cells (PBMC) to lypopolysaccharides (LPS) has been investigated in our laboratories for a long time. In particular, a possible correlation between blastogenic response to LPS and disease resistance of periparturient dairy cows had been observed in previous studies. Most important, low responder cows presented a higher frequency of disease cases after calving, compared with high responder animals. Owing to the above, different aspects of the blastogenic response to LPS were investigated on PBMC of healthy Friesian cows, using a 72-hour Bromodeoxyuridin (BrDU) cell proliferation assay. Stimulation with LPS induced little if any replication of bovine PBMC over 72 hours despite consistent BrDU detection in all the PBMC samples under study. Poor replication of LPS-stimulated PBMC was confirmed by cell cycle and cell growth flow cytometry analyses. In particular, LPS stimulation gave rise to very low percentages of S phase cells, sometimes lower than in control, unstimulated cells, as opposed to Concanavalin A-stimulated PBMC. Magnetic separation and analysis of BrDU-treated bovine PBMC after exposure to LPS showed that both B and CD4 T cells are involved in the blastogenic response to LPS, in contrast with current data based on human and murine models. Finally, LPS caused an early, specific up-regulation of TNF-α and TLR4 genes in bovine PBMC, and significant correlations were shown between the expression of inflammatory cytokine and Indoleamine-pyrrole 2,3-dioxygenase (IDO1) genes. On the whole, our data indicate that differences in the blastogenic response to LPS could be partly accounted for by heterogenicity of responding cells (B and T lymphocytes), which might also have an impact on induction and regulation of inflammatory responses and endotoxin tolerance.