Immune Response In Dogs Research Articles

Evaluation of safety and immunogenicity of a genetically modified rabies virus for use as an oral vaccine in several non-target species1

Oral immunization is an alternative or supplementary approach that can significantly improve dog vaccination coverage, especially for free-roaming dogs. Safe and effective oral rabies vaccines for dogs are still being sought. In our previous studies, we generated a genetically modified rabies virus (RABV) ERA strain, rERAG333E, containing a mutation from arginine (Arg, R) to glutamic acid (Glu, E) at residue 333 of the G protein (G333E). Our previous results demonstrated that rERAG333E was safe for adult mice and dogs, and oral vaccination with rERAG333E induced a strong and long-lasting protective immune response in dogs. Here, we further investigated the safety and immunogenicity of rERAG333E in non-target species, including suckling mice, rhesus monkeys, foxes, raccoon dogs, piglets, goats, and sheep. Suckling mice studies demonstrated that the G333E mutation significantly reduced the virulence of the ERA strain. All of the suckling mice aged 10 days and above survived and showed no apparent signs of disease after intracerebral inoculation with rERAG333E. Animal studies demonstrated that rERAG333E was safe in rhesus monkeys, foxes, raccoon dogs, piglets, goats, and sheep. None of those animals inoculated orally with 10 times the intended field dose of rERAG333E showed abnormal clinical signs before and after the booster immunization with Rabvac 3, an inactivated rabies vaccine. Meanwhile, oral inoculation with rERAG333E induced strong neutralizing antibody (NA) responses to RABV in rhesus monkeys, foxes, raccoon dogs, and piglets. These results demonstrated that rERAG333E has the potential to serve as a safe oral rabies vaccine for dogs.

Betaine and L-Carnitine Synergistically Influence the Metabolome and Immune Response in Dogs

This study used thirty-two dogs, which were assigned to a preferred period of 14 days and then assigned to one of the four treatment foods: control (containing no added betaine, no added L-carnitine), control with 0.5% added betaine (Treatment 2), control with no added betaine and 300 ppm added L-carnitine (Treatment 3), or control with 0.5% added betaine and 300 ppm added L-carnitine (Treatment 4). All treatment foods were fed for ninety days. Untargeted blood metabolomic analysis and immune response were measured at the beginning and end of the 90-day feeding trial. Feeding betaine increased single-carbon metabolites while decreasing many carnitine-containing metabolites. Feeding L-carnitine increased many carnitine metabolites, while the combination synergistically influenced the metabolome. The combination of betaine and L-carnitine increased the cytokines released in a Tru-culture system in response to stimulation while numerically decreasing their release when unstimulated. Therefore, the combination of dietary betaine and L-carnitine could have the dual positive effects of reducing cytokine stimulation, controlling inflammation during health, and providing a robust response to bacterial infection.

Long-term immunity induced by SPBN GASGAS in orally vaccinated dogs is non-inferior to inactivated rabies vaccines

In a long-term immunogenicity study (1100 days post vaccination) in local Thai dogs the immune response of the oral rabies vaccine SPBN GASGAS was compared to those elicited by a commercial inactivated vaccine using immunobridging. Based on the detection of rabies virus binding (rVBA) and rabies virus neutralizing antibodies (rVNA) as measured by ELISA and Rapid Fluorescent Focus Inhibition Test (RFFIT) the long-term immune response in dogs vaccinated orally with the SPBNA GASGAS strain of rabies vaccine in a bait was non-inferior to a conventional inactivated rabies vaccine. The outcome of this study supports extending the originally claimed duration of immunity (DOI) of SPBN GASGAS after oral vaccination for dogs from 6 to 30 months.

PSVIII-19 Saccharomyces Cerevisiae Fermentation Product Modulates Blood Cell Counts and Gut Immunity in Healthy Adult Cats

Abstract Dietary Saccharomyces cerevisiae fermentation product (SCFP) has been shown to modulate immune responses in dogs and livestock. Information about feeding SCFP to cats, however, is lacking. This study aimed to evaluate the effects of SCFP (TruMune, Cargill, Inc.) on immune indices including fecal IgA, serum oxidative stress markers, and toll-like receptor (TLR) responsiveness in healthy adult cats. Sixty-three cats (mean age = 6.3 ± 2.8 yr; mean BW = 4.4 ± 0.1 kg) were used in a randomized complete block design. All cats were fed a control kibble diet without SCFP (CON) during a 21-day adaptation period. Cats were randomly assigned to one of the three diets including CON, a dry diet containing 150 mg/kg BW SCFP (TRUL), and a dry diet containing 300 mg/kg BW SCFP (TRUH) and fed for 42 days. Fecal samples were collected on days 0, 21, and 42 for fecal IgA analysis. Serum samples were collected on days 0 and 42 for measurements of serum chemistry, complete blood count, and oxidative stress markers. Whole blood samples were collected on day 42 for TLR responsive evaluation. Protocols were approved by the facility’s Institutional Animal Care and Use committee before the study. Data were analyzed using R v.4.1.2. Differences P < 0.05 were considered significant and P < 0.10 as trends. All serum chemistry and blood cell count values were within the reference ranges, with serum chemistry not affected by SCFP. Absolute cell counts of eosinophils and neutrophils decreased linearly with SCFP dose (P < 0.001) while absolute cell counts of lymphocytes and monocytes increased linearly with SCFP dose (P < 0.01). TLR responsiveness was not altered (P > 0.05) by SCFP. A tendency (P = 0.08) of quadratic treatment dose response was noted for thiobarbituric acids from the thiobarbituric acid reactive substance assay, with cats fed TRUL having the greatest thiobarbituric acid values. Fecal IgA showed a quadratic treatment dose response on day 42 (P = 0.01), with cats fed TRUL having the lowest fecal IgA values. These results demonstrate that SCFP may affect immune outcomes including blood cell counts and fecal IgA in adult healthy cats.

Development of HPV16 mouse and dog models for more accurate prediction of human vaccine efficacy

BackgroundAnimal models are essential to understand the physiopathology of human diseases but also to evaluate new therapies. However, for several diseases there is no appropriate animal model, which complicates the development of effective therapies. HPV infections, responsible for carcinoma cancers, are among these. So far, the lack of relevant animal models has hampered the development of therapeutic vaccines. In this study, we used a candidate therapeutic vaccine named C216, similar to the ProCervix candidate therapeutic vaccine, to validate new mouse and dog HPV preclinical models. ProCervix has shown promising results with classical subcutaneous murine TC-1 cell tumor isografts but has failed in a phase II study.ResultsWe first generated E7/HPV16 syngeneic transgenic mice in which the expression of the E7 antigen could be switched on through the use of Cre–lox recombination. Non-integrative LentiFlash® viral particles were used to locally deliver Cre mRNA, resulting in E7/HPV16 expression and GFP reporter fluorescence. The expression of E7/HPV16 was monitored by in vivo fluorescence using Cellvizio imaging and by local mRNA expression quantification. In the experimental conditions used, we observed no differences in E7 expression between C216 vaccinated and control groups. To mimic the MHC diversity of humans, E7/HPV16 transgenes were locally delivered by injection of lentiviral particles in the muscle of dogs. Vaccination with C216, tested with two different adjuvants, induced a strong immune response in dogs. However, we detected no relationship between the level of cellular response against E7/HPV16 and the elimination of E7-expressing cells, either by fluorescence or by RT-ddPCR analysis.ConclusionsIn this study, we have developed two animal models, with a genetic design that is easily transposable to different antigens, to validate the efficacy of candidate vaccines. Our results indicate that, despite being immunogenic, the C216 candidate vaccine did not induce a sufficiently strong immune response to eliminate infected cells. Our results are in line with the failure of the ProCervix vaccine that was observed at the end of the phase II clinical trial, reinforcing the relevance of appropriate animal models.

Genetic haplotypes associated with immune response to Leishmania infantum infection in dogs.

Leishmaniasis is a zoonotic parasitic disease, and the main reservoir of the parasite is the dog, although recent years have seen an increase in other mammalian species. In the Mediterranean region, where it is an endemic disease, it is caused by the species Leishmania infantum. The Ibizan hound, an autochthonous breed of this region, appears to have a genetic resistance to parasitic infection, whereas other canine breeds, such as the Boxer, are susceptible to infection. These differences are related to the differentiated activation of the immune response, with the Ibizan hound activating the Th1 immune response, whereas the Boxer breed triggers the Th2 immune response. Cytokine levels and genomic haplotypes of several genes involved in the immune response were analysed in twenty-eight Ibizan hound (resistant canine breed model) and twenty-four Boxer (susceptible canine breed) without clinical signs in the Mediterranean region. Cytokine levels were analysed by ELISA commercial kits and haplotypes were studied using CanineHD DNA Analysis BeadChip including 165,480 mapped positions. The results show 126 haplotypes associated with differential immune response in dogs. Specifically, haplotypes in IL12RB1, IL6R, CIITA, THEMIS, NOXA1, HEY2, RAB38, SLC35D2, SLC28A3, RASEF and DAPK1 genes are associated with serum levels of IFN-γ, IL-2, IL-8, and IL-18. These results suggest that the resistance or susceptibility to Leishmania infantum infection could be a consequence of haplotypes in several genes related to immune response. Future studies are needed to elucidate the relationship of these haplotypes with immune response and gene expression regulation.

Evaluation of Host Constitutive and Ex Vivo Coccidioidal Antigen-Stimulated Immune Response in Dogs with Naturally Acquired Coccidioidomycosis.

The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105-310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations (p = 0.02) and serum CRP concentrations compared to controls (p < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination (p = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α (p = 0.0003), interleukin (IL)-6 (p = 0.04), interferon (IFN)-γ (p = 0.03), monocyte chemoattractant protein (MCP)-1 (p = 0.02), IL-10 (p = 0.02), and lower IL-8 (p = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis.

Dietary yeast beta 1,3/1,6 glucan supplemented to adult Labrador Retrievers alters peripheral blood immune cell responses to vaccination challenge without affecting protective immunity.

Yeast-derived 1,3/1,6 beta-glucans may alter host immunity to produce robust and quickly resolved responses that align with companion animal health goals. In adult dogs, immunomodulation by yeast 1,3/1,6 beta-glucans in extruded kibble diet have not been well-documented. The study objective was to evaluate systemic immune responses in dogs fed kibble diets with two yeast 1,3/1,6 beta-glucans doses before and after vaccine challenge. Twenty-four adult Labrador Retrievers were assigned to 3 dietary treatments consisting of a basal diet (control) supplemented with 0.012 or 0.023% (0.5 or 1X, respectively) yeast 1,3/1,6 beta-glucan with equal sex representation within each treatment (8 dogs/diet). Animals were fed experimental diets for a 29d acclimation period, after which baseline blood samples were collected before administration of a combination canine distemper virus, parvovirus, and adenovirus-2 vaccine. Blood samples were collected weekly for 21d following vaccination with whole blood for CBC analysis, serum for titer and cytokine assays, and peripheral blood mononuclear cells (PBMC) isolated for flow cytometric immune cell profiling. Data were analyzed using the MIXED procedure with diet and timepoint fixed effects. Serum titer was analyzed by Kruskal-Wallis test (SAS 9.4; P≤0.05). Prior to vaccination, beta-glucan diets did not affect serum cytokines, antibody titer, or immune cell populations. In the first 7 days post-vaccination (dpv), PBMC CD21 low B cells increased in 36.5-58.1% in all groups but the magnitude of change was lesser in the 0.5X beta-glucan diet resulting in 25.6% lower CD21 low populations compared to control-fed dogs (P=0.007). By 21dpv, B cell populations recovered to baseline levels in dogs fed 1X beta-glucan, but CD21 high cells remained elevated 50.5% in dogs fed 0.5X beta-glucan diets compared to baseline (P<0.0001). While no differences in serum titer or cytokines were observed, feeding both beta-glucan diets maintained stable blood monocytes whereas a 53.0% decrease between baseline and 14dpv was observed in control-fed dogs (P=0.01). Collectively, these outcomes suggest that a 1X dose of 1,3/1,6 yeast beta-glucan in extruded kibble diets altered monocytes associated with trained immunity, did not reduce PBMC CD21 low B cell responsiveness, and simultaneously contributed to B cell population resolution by 21dpv in adult dogs. Additional research to assess the functionality of these changes is needed.

Phenotypic and Transcriptional Changes of Pulmonary Immune Responses in Dogs Following Canine Distemper Virus Infection.

Canine distemper virus (CDV), a morbillivirus within the family Paramyxoviridae, is a highly contagious infectious agent causing a multisystemic, devastating disease in a broad range of host species, characterized by severe immunosuppression, encephalitis and pneumonia. The present study aimed at investigating pulmonary immune responses of CDV-infected dogs in situ using immunohistochemistry and whole transcriptome analyses by bulk RNA sequencing. Spatiotemporal analysis of phenotypic changes revealed pulmonary immune responses primarily driven by MHC-II+, Iba-1+ and CD204+ innate immune cells during acute and subacute infection phases, which paralleled pathologic lesion development and coincided with high viral loads in CDV-infected lungs. CD20+ B cell numbers initially declined, followed by lymphoid repopulation in the advanced disease phase. Transcriptome analysis demonstrated an increased expression of transcripts related to innate immunity, antiviral defense mechanisms, type I interferon responses and regulation of cell death in the lung of CDV-infected dogs. Molecular analyses also revealed disturbed cytokine responses with a pro-inflammatory M1 macrophage polarization and impaired mucociliary defense in CDV-infected lungs. The exploratory study provides detailed data on CDV-related pulmonary immune responses, expanding the list of immunologic parameters potentially leading to viral elimination and virus-induced pulmonary immunopathology in canine distemper.

Canine Cytokines Profile in an Endemic Region of L. infantum: Related Factors.

Canine leishmaniosis is caused by infection with parasite Leishmania infantum, which are transmitted by sandflies Phlebotomus. Canine leishmaniosis is an endemic disease in the Mediterranean region. The immune response could vary between hosts and determines the severity of the disease and clinical features. The aim of this study was to analyze the serum levels of cytokines TNF-α, IFN-γ, IL-2, IL-6, and IL-8, which are related to the activation of Th1 or Th2 immune responses in dogs living in the L. infantum endemic region. Moreover, we intend to relate and correlate these levels with different factors, such as sex, age, diet, lifestyle, and breed. Epidemiological data and serum were recovered for seventy-eight dogs, and serum levels of cytokines described previously were analyzed by using the ELISA method. The results showed differences in serum levels of IFN-γ, IL-2, and IL-8 between breeds. The lifestyle also affected serum levels of IL-2. The main conclusion of this study is that Ibizan hounds and crossbred dogs have a serological profile of cytokines that seems to indicate certain protections against infection by L. infantum compared to boxer and purebred breeds.

Immune Response After Rabies Vaccination in Owned Free-Roaming Domestic Dogs in Flores Island, Indonesia.

Vaccination is the main tool to prevent the circulation of rabies in dog populations. The development of an immune response after vaccination differs between individual dogs and depends on many factors such as dog characteristics, management, or genetics. Here, we first investigated the level of, and associated factors for, the presence of binding antibodies in 130 healthy dogs from Flores Island, Indonesia. Secondly, we identified factors associated with the development of binding antibodies within 30 days after vaccination among a subsample of dogs that had a binding antibody titre <0.5 EU/ml at the day of vaccination (D0, N = 91). Blood samples were collected from the individual dogs immediately before vaccination at D0 and 30 days after vaccination (D30). The rabies antibody titres were determined using ELISAs. Information on potential risk factors such as the dog's age and sex, history of vaccination, type and frequency of feeding, and BCS (body condition score) were gathered during interviews at D0. Regression analyses were performed to identify the risk factors associated with the presence of binding antibody titre ≥0.5 EU/ml at D0 for the 130 dogs and the development of binding antibody titre ≥0.5EU/ml at D30 for the 91 dogs. The results showed that the proportion of dogs with antibody titre ≥0.5 EU/ml was 30% (39/130) at D0. The only factors found to be significantly influencing the presence of binding antibodies titres ≥0.5 EU/ml was previous vaccination within 1 year before D0 [46.8 vs. 14.7%, Odds ratio (OR) = 3.6, 95%CI 1.5–9.3; p-value = 0.006], although the same trend was found for dogs of higher age and better BCS. Eighty-six percent (79/91) of dogs whose rabies binding antibody level was <0.5 EU/ml at D0 had developed an adequate immune response (≥0.5 EU/ml) at D30. Almost a significantly higher proportion developed an adequate immune response in dogs of good BCS compared to those of poor BCS (95.3% vs. 79.2%, OR = 4.7, 95%CI 1.1–32.5; p-value = 0.057. Twelve (13.2%) dogs retain binding antibody level <0.5 EU/ml at D30, indicating poor immune response after vaccination. A majority of them did not receive vaccine before D0 according to the owner and had poor BCS (83.3%; 10/12). Our findings show the high effectiveness of rabies vaccine in under field conditions to develop measurable immunity and the importance of a good BCS, often achievable by good dog keeping conditions, for developing efficient immunity after parenteral vaccination in dogs.

Activation of Canine, Mouse and Human TLR2 and TLR4 by Inactivated Leptospira Vaccine Strains.

Canine Leptospira vaccines contain inactivated strains of pathogenic Leptospira, the causative agents of leptospirosis. For an effective response to vaccination, activation of the innate immune system via pattern recognition receptors such as TLRs is crucial. However, it is not known which TLRs are activated by Leptospira in dogs. To investigate the involvement of canine TLR2, TLR4, and TLR5 in the recognition of Leptospira, we stimulated canine moDC and reporter cells expressing canine TLR2 with either whole-inactivated bacteria or purified LPS of Leptospira strains, representing the serogroups generally used in canine leptospirosis vaccines. Using the endotoxin neutralizing reagent polymyxin B and TLR4 antagonist RS-LPS, we demonstrate that Leptospira LPS and canine TLR4 are involved in IL-1β production as well as in the uptake of inactivated Leptospira in canine moDC. Furthermore, polymyxin B only partially inhibited IL-1β production induced by inactivated Leptospira, suggesting that next to TLR4, also other TLRs may be involved. The observed activation of canine TLR2-expressing reporter cells by inactivated Leptospira strains indicates that TLR2 could be one of these TLRs. Next, we analyzed TLR2 and TLR4 activating capabilities by the same Leptospira strains using human and mouse TLR-expressing reporter cells. Inactivated Leptospira and leptospiral LPS activated not only mouse, but also human TLR4 and this activation was shown to be LPS dependent in both cases. Additionally, inactivated Leptospira activated mouse and human TLR2-expressing reporter cell lines. In our study, we could not identify significant species differences in the recognition of Leptospira by TLR2 and TLR4 between dog, human and mouse. Lastly, we show that these inactivated Leptospira strains are recognized by both mouse and human TLR5 reporter cells only after exposure to additional heat-treatment. Unfortunately, we were not able to confirm this in the canine system. Our data show that TLR2 and TLR4 are involved in the recognition of Leptospira strains used in the production of canine Leptospira vaccines. This study contributes to the understanding of Leptospira-induced innate immune responses in dogs, humans, and mice. Future studies are needed to further explore the role of canine TLR2, TLR4 and TLR5 in the induction of vaccine-mediated immunity against Leptospira.

Abstract 1693: Multi-national, multi-center collaboration to develop a novel gene expression tool for comparative translational immuno-oncology

Abstract Harnessing the immune system to eliminate cancer and prevent its recurrence is proving to be a powerful therapeutic approach that has achieved unprecedented successes in hematological malignancies and some malignancies with a high tumor burden. Next generation immune therapies to improve clinical responses and widen the reach of immunotherapy are being designed at a rapid pace, and informative pre-clinical testing of these approaches can be greatly facilitated using immune competent animals with spontaneous tumors. Pet dogs are immunologically outbred, immune competent and develop spontaneous tumors such as non-Hodgkin's lymphoma, glioblastoma, osteosarcoma, urothelial carcinoma and melanoma that share remarkable clinical, biological and genetic features with their human counterparts. As such, pre-clinical testing of immune therapeutic approaches in dogs with cancer promises to accurately inform human clinical trial design. For this comparative approach to provide maximum information to accelerate human clinical translation of next generation immunotherapies and identify correlative biomarkers of therapeutic response, it is necessary to develop research tools for deep interrogation of the canine immune response. Here we present work conducted through a year-long, multi-center, global collaboration resulting in the creation of a novel gene expression tool for studies of the immune response in dogs treated with immuno-oncology and targeted therapies. This original approach utilizes the NanoString's nCounter® platform and is termed the Canine IO Panel, with inclusion of 800 canine genes for studying the immune response. The panel can be used with tissue, blood or other biospecimens and represents a companion panel to the widely recognized nCounter Human IO 360™ panel currently in use with human clinical trials. The customizable panel segments genes into 8 core components including Cytokine &amp; Chemokine Signaling, Interferon Signaling, Checkpoint Signaling, Complement Cascade, Immune Cell Abundance, Tumor Immunogenicity, Inhibitory Tumor Mechanisms, and Stromal Factors. Genes were selected based on their relevance for the study of oncology as well as expression profiles from both RNA-Seq and nCounter experiments. Additionally, the canine reference transcriptome, based on CanFam3.1 was utilized for designing the probes; the known genomic variability of dogs in addition to the nCounter hybridization chemistry result in compatibility across a variety of breeds. This new Canine IO panel, used in conjunction with the nCounter platform and correlated with the abundance of data that exist on the platform for similar human IO studies, creates a powerful tool for researchers undertaking comparative human and veterinary studies aiming to develop and improve the understanding and treatment of both canine and human cancers. Citation Format: Nicola Mason, Christina Bailey, Erin Piazza, Alexander F. Haake, Achim D. Gruber, Cheryl London, M. R. Chambers, Steven W. Dow, G. Elizabeth Pluhar, Alina K. Langenhagen, Dhawan Deepika, Deborah W. Knapp, Qi Long, Robert B. Rebhun. Multi-national, multi-center collaboration to develop a novel gene expression tool for comparative translational immuno-oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1693.

Nonhypoalbuminemic Inflammatory Bowel Disease in Dogs as Disease Model.

The incidence of inflammatory bowel disease (IBD) is increasing worldwide, emphasizing the need of relevant models, as dogs spontaneously affected by IBD may be, for better knowledge of the disease's physiopathology. We studied 22 client-owned dogs suffering from IBD without protein loss and 14 control dogs. Biopsies were obtained from the duodenum, ileum, and colon. Inflammatory grade was assessed by histopathology, immunohistochemistry, and chemokine analysis. The expression of Toll-like receptors (TLR) in mucosa was immunohistochemically evaluated. Antibody levels against bacterial ligands (lipopolysaccharide [LPS] and flagellin) were measured in sera using enzyme-linked immunoassay. Dogs with IBD showed low to severe clinical disease. Histopathologically, the gut of dogs with IBD did not exhibit significant alterations compared with controls except in the colon. The number of CD3+ T lymphocytes was decreased in the ileum and colon of dogs with IBD compared with controls, whereas the numbers of Foxp3+, CD20+, and CD204+ cells were similar in the 2 groups. Three chemokines, but no cytokines, were detected at the protein level in the mucosa, and the disease poorly affected their tissue concentrations. Dogs with IBD exhibited higher serum reactivity against LPS and flagellin than controls but similar immunoreactivity against the receptors TLR4 and TLR5. In addition, TLR2 and TLR9 showed similar expression patterns in both groups of dogs. Our data described dysregulated immune responses in dogs affected by IBD without protein loss. Despite fairly homogeneous dog cohorts, we were still faced with interindividual variability, and new studies with larger cohorts are needed to validate the dog as a model.

Evaluation of Cellular Immune Responses in Dogs Immunized with Alum-Precipitated Autoclaved Leishmania major along with BCG and Imiquimod.

Background:We aimed to investigate the potential effects of BCG and imiquimod on improvement of current experimental L. major vaccine against dogs in an endemic area of Zoonotic visceral leishmaniasis (ZVL) in Iran.Methods:During 2012 till 2014, seven mixed-breed shepherd dogs with no anti-Leishmania antibodies and no response to Leishmanin reagent were immunized with 2 doses of alum-precipitated autoclaved L. major (Alum-AML) while BCG and imiquimod (for skin pre-treatment) were used as adjuvants. The productions of a few characteristic cytokines of T-helper immune responses and the development of delayed-type hypersensitivity (DTH) of the immunized animals were then evaluated, up to 300 days. Blood samples were collected at 0, 30, 80 and 300 d post-vaccination and the concentrations of IFN-γ, IL10, IL-12 and TGF-β cytokines secreted from PBMCs at these time-points were quantified by ELISA. DTH was evaluated by Leishmanin skin test (LST).Results:Although a similar LST conversion was observed at all time-points, the cytokine measurement results indicated significantly higher levels of IFN-γ at day 80 and elevated levels of IL-10 at days 80 and 300, post-vaccination. Moreover, a significantly higher IFN-γ/IL-10 ratio was observed at day 30 post-vaccination compared to the other time-points.Conclusion:Although a Th1-like response could be observed at day 30 post-vaccination, the development of cytokine profiles was inclined toward mixed Th1 and Th2 responses at days 80 and 300 post-vaccination. This situation may indicate the requirement of an additional boosting by this Alum-AML formula, in order to induce long-lasting protection against ZVL.

A highly efficient recombinant canarypox virus-based vaccine against canine distemper virus constructed using the CRISPR/Cas9 gene editing method

Canine distemper virus (CDV) is the causative agent of canine distemper (CD), which is one of the most important infectious diseases affecting wild and domestic carnivores. Vaccination represents an effective approach to prevent CDV infection among domestic carnivores. Canarypox-vectored recombinant CD vaccines (such as Recombitek CDV, PureVax Ferret Distemper, and Merial) with the CDV hemagglutinin (H) and fusion (F) genes can induce a potent immune response in dogs and ferrets. However, the vaccine’s effectiveness varies with the species. In the current study, we developed a highly efficient recombinant canarypox virus termed as “ALVAC-CDV-M-F-H/C5−” that contained CDV virus-like particles (VLPs) by using the CRISPR/Cas9 gene editing method, which enabled concurrent expression of the matrix (M), H, and F genes. The recombinant strain provided faster seroconversion than the parent strain among minks as well as provided higher rates of antibody positivity than the parent strain among foxes and minks even before the administration of a second booster vaccination. We demonstrated, for the first time, that the CRISPR/Cas9 system can be applied for the rapid and efficient modification of the ALVAC-CDV-F-H genome and also that a high-dose new recombinant strain that produces CDV VLPs may present good outcomes in the prevention of CD among foxes and minks.

Toll-Like Receptors 2, 4, and 7, Interferon-Gamma, Interleukin 10, and Programmed Death Ligand 1 Transcripts in Leishmanin Skin Test-Positive Reactions of Ibizan Hound Dogs.

The leishmanin skin test (LST) is an in vivo technique commonly used to evaluate the Leishmania-specific cellular immune response in dogs. However, information regarding the local immune response in LST-positive reactions is scarce. We examined the pattern of toll-like receptor 2 (TLR2), TLR4, TLR7, interleukin- (IL-) 10, interferon gamma (IFN-γ), and (program death ligand) PD-L1 gene expression in LST-positive reactions and paired normal-looking skin of nine infected Ibizan hound dogs. Healthy skin from ten seronegative dogs from a nonendemic area was analysed as a negative control. Immune gene expressions were examined by quantitative PCR (qPCR) analysis. LST-positive reactions presented significant upregulation of TLR2, TLR4, IL-10, IFN-γ, and PD-L1 and downregulation of TLR7 when compared with healthy skin of seronegative control dogs from a nonendemic area. All transcripts but TLR7 were significantly higher in LST-positive reaction than in paired normal-looking skin of Ibizan hound. The expression profile of immune genes in LST-positive reactions was similar to that previously observed in clinically lesioned skin of mildly diseased dogs with papular dermatitis due to Leishmania infantum infection. This data provide additional support for the important role of TLRs in canine leishmaniosis.

Quantification of immuno-regulatory cytokine and toll-like receptors gene expression in dogs with generalized demodicosis

The proliferation of Demodex mites is mainly controlled by host immunity; however, the precised mechanism of host–mite interplay and host immune response in the cutaneous microenvironment of dogs with generalized demodicosis (GD) are not yet established. In the present study, we envisaged the alterations in the expression of toll-like receptors (TLRs) and immuno-regulatory cytokine gene in the skin lesions and peripheral blood mononuclear cells (PBMCs) of dogs with GD. The expression of TLR2, TLR6, IFN-γ, TGF-β and IL-10 genes in the skin lesions and PBMCs of 15 dogs with GD was quantified by qRT-PCR. Compared to healthy dogs, significantly elevated expression of TLR2 (P = 0.048), TGF-β (P = 0.04) and IL-10 (P = 0.012) were found in the PBMCs of dogs with GD. Conversely, there was significantly reduced expression of TLR6 gene (P = 0.021) in the PBMCs of these dogs. The infested dogs also revealed significantly elevated expression of TLR2 gene (P = 0.034) in the skin lesions, while, the expression of the TLR6 gene was found to be significantly (P = 0.004) reduced. Interestingly, significant alterations in TGF-β (P = 0.105) and IL-10 (P = 0.162) genes expression were not observed in the skin lesions of diseased dogs. Our findings suggest that Demodex mites contribute to a different systemic and cutaneous immune response in dogs for their proliferation, and consequently the development of GD. Therefore, Demodex mites might be inducing the immunosuppression through activating the systemic over-expression of immunosuppressive cytokines; however, in the cutaneous lesions, the expression of immunosuppressive cytokines remained unaltered. Both systemic and local over-expression of TLR2 and reduced expression of TLR6 genes might be responsible for the inflammatory signs of canine demodicosis and helping to the mite to escape the host immunity.

Combined in vitro IL-12 and IL-15 stimulation promotes cellular immune response in dogs with visceral leishmaniasis.

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.

Dogs with osteosarcoma have altered pro‐ and anti‐inflammatory cytokine profiles

BackgroundCurrent advances in immunotherapy are an exciting area of study in canine osteosarcoma (OSA). The objective of this study was to determine the immune response in dogs with osteosarcoma by measuring stimulated leukocyte production of tumor necrosis factor (TNF), interleukin (IL)‐6, IL‐10 and TNF and IL‐6 to IL‐10 ratios.MethodsWhole blood was collected from dogs with osteosarcoma receiving non‐steroidal anti‐inflammatory drugs (NSAIDs, n = 11), dogs with osteosarcoma not receiving NSAIDs (n = 14) and healthy dogs (n = 5).ResultsNo difference in TNF production was found among healthy and OSA dogs regardless of NSAID administration following stimulation with lipopolysaccharide (LPS) (p = .410), lipoteichoic acid (LTA) (p = .693) or PBS (p = .120). Leukocyte IL‐6 production was greater in all dogs with OSA after stimulation with LPS (p = .015), LTA (p = .014) and PBS (p = .034) with no difference between OSA dogs receiving NSAIDs and those not. No differences in IL‐10 were found among healthy controls and dogs with OSA regardless of NSAID use. There was no difference among groups for LPS‐stimulated TNF to IL‐10 ratios (p = .407). For LTA‐stimulated leukocytes, the TNF to IL‐10 ratio was lower in dogs with OSA than in healthy dogs (p = .031) with no difference between OSA NSAID dogs compared to OSA non‐NSAID dogs (p = .059). No differences were found in LPS (p = .310)‐ or LTA (p = .265)‐stimulated leukocyte IL‐6 to IL‐10 production ratios among groups.ConclusionsDogs with osteosarcoma have an altered pro‐ and anti‐inflammatory immunologic profile compared to healthy dogs regardless of NSAID use. Further study is indicated to determine the potential prognostic and therapeutic implications of these findings.