Search

The detection of Leishmania tarentolae in sympatric areas where Leishmania infantum is endemic raised questions regarding the protective effect exerted in dogs by L. tarentolae when in coinfection. This study aimed monitoring the in vitro gene expression of pro- (IFN- γ; TNF-α; IL-12) and anti-inflammatory (IL-4; IL-6; IL-10) cytokines in primary canine macrophages infected by L. tarentolae and L. infantum in single and in co-infections. Macrophages differentiated from dog blood mononuclear cells were infected with the L. tarentolae field-isolated (RI-325) and laboratory (LEM-124) strains, with L. infantum laboratory strain (IPT1), or both. Infection and the number of amastigotes per infected cell were evaluated microscopically by counting a total of 200 cells between 4 and 96 h. Cytokine gene expression was analyzed by real-time PCR from infected macrophages mRNA. Single infections presented higher expression of the cytokines IL-4 and IL-6, and lower of IL-12. Co-infections induced a lower gene expression of IL-4 and IL-6, and a higher gene expression of IL-12, correlating with the low amastigote burden despite the slight increase of infected cells. Data highlight the potential protective effect of L. tarentolae against L. infantum in co-infection by the reduced anti-inflammatory and increased pro-inflammatory cytokines gene expression, opening new perspectives for a canine vaccine development exploiting the non-pathogenic L. tarentolae., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Leishmaniasis is a significant public health concern, with dogs as the primary reservoir in urban scenarios and facilitating transmission. Diagnosing infected dogs is a crucial step for public health interventions, and the development of new diagnostic platforms can significantly enhance efforts in various regions worldwide. Given the limited availability of diagnostic methods in Colombia, this study evaluates the effectiveness of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) based on the recombinant protein rLicNTPDase-2 to detect Leishmania in infected dogs. Serum samples were collected from dogs in both endemic and non-endemic areas and classified as natural standards based on prior parasitological diagnoses. The results revealed 24 true positives (TP) and 9 true negatives (TN). Subsequently, the test was then validated with samples from symptomatic and asymptomatic animals, alongside the standards, yielding a specificity of 96 %, a sensitivity of 81 %, efficiency of 90.6 %, a positive predictive value of 92.8 %, and a negative predictive value of 89.6 %. The positive likelihood ratio (RV+) was 20, while the negative likelihood ratio (RV-) was 0.19, indicating high relevance and a robust clinical utility. The area under the curve (AUC) was 1.00, suggesting that the test has excellent discriminatory ability, significantly deviating from the reference diagonal. This is further supported by the significant difference(p < 0.0001) between TN and TP results determined by Fisher's exact test. Involving 163 animals showed 47 % positive and 46 % negative results with a significant difference (p < 0.05) in the mean optical density (OD) values between positive and negative samples. These findings indicate that the ELISA test effectively differentiates between positive and negative samples based on OD values. This study suggests that ELISA based on the recombinant antigen rLicNTPDase-2 could serve as a viable alternative for the serodiagnosis of leishmaniasis in canines in Colombia., Competing Interests: Declaration of competing interest There are no competing interests that may influence the results of this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cutaneous and subcutaneous mast cell tumors (MCTs) are common canine neoplasms characterized by variable biological behavior. Tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) can be effective prognostic markers in numerous human neoplasms and are increasingly investigated in dogs. The aim of this study was to characterize immune cells in canine MCTs and their relationship with histological location (cutaneous, subcutaneous) and histologic nodal metastatic status (HN0-3). Thirty-eight MCTs (26 cutaneous, 12 subcutaneous) from 33 dogs with known sentinel lymph node (SLN) metastatic status were immunolabeled for Iba1 (macrophages), CD20 (B cells), CD3 (T cells), and Foxp3 (regulatory T cells). Semiquantitative scoring of interstitial and perivascular CD3+, CD20+, and Foxp3+ cells and morphological evaluation of Iba1+ cells were performed. For each marker, the percent immunopositive area was evaluated by image analysis. All MCTs were diffusely infiltrated by Iba1+ cells and variably infiltrated by CD20+, CD3+, and rare Foxp3+ cells. Stellate/spindle Iba1+ cells were associated with HN2 and HN3 SLNs. Perivascular Foxp3+ cells, CD3+ cells, and percent CD3+ areas were increased in subcutaneous MCTs. Interstitial CD3+ cells were increased in cutaneous MCTs with HN0 SLNs. No differences in CD20+ cells were identified between cutaneous and subcutaneous MCTs and among SLN classes. MCTs were markedly infiltrated by TAMs and variably infiltrated by TILs. Stellate/spindle morphology of TAMs associated with HN2 and HN3 SLNs is suggestive of a pro-tumoral (M2) phenotype. Cutaneous and subcutaneous MCTs have different tumor-immune microenvironments, and T-cell infiltration might contribute to prevention of nodal metastatic spread of cutaneous MCTs., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Chronic lymphoid leukemia (CLL) is a hematological disorder characterized by the clonal expansion of small mature lymphocytes that accumulate in the blood and bone marrow. CLL can arise from B-, T-, or natural killer cell clones. The cytological evaluation of blood smears is often the simplest and least invasive method for diagnosing lymphoid leukemia. Immunophenotyping is used to further subclassify the type of lymphoid leukemia., Case Presentation: A 15-year-old, 4.4-kg spayed female Shih Tzu was presented to the veterinary medical teaching hospital of Kangwon National University. Despite having a normal appetite and activity level, cervical and inguinal lymph node enlargement was noted on physical examination. Complete blood count revealed severe leukocytosis, severe lymphocytosis, and monocytosis. Splenomegaly, hepatomegaly, and lymph node enlargement were detected on radiographic and ultrasonographic examination. Immunophenotyping was performed using peripheral blood mononuclear cells (PBMCs). The majority of lymphocytes exhibited the following profiles: CD3 - CD79a - (97.5%), CD4 - CD8 - (98.6%), CD21 - CD79a - (98.4%), CD34 - (0.1%), CD45 + (99.6%), major histocompatibility complex class II + (99.5%), and CD14 - (0.5%). Based on the immunophenotyping results, possible differentials considered included the following: the majority of lymphocytes may be natural killer (NK) cell clones, plasma cell clones, or show aberrant expression or loss of CD21 marker due to the neoplastic nature of the cells. Further flow cytometry was performed using antibodies against CD3, CD5, CD94, and granzyme B. The combined results indicated that the predominant lymphocyte subset in the PBMCs was CD3 - CD5 - CD21 - CD94 - granzyme B - . To confirm monoclonality and exclude the aberrant loss of CD markers, a polymerase chain reaction for antigen receptor rearrangement (PARR) assay was conducted. The PARR assay, using DNA from blood and lymph node samples, showed B-cell monoclonality. Immunocytochemistry using PBMCs showed that the plasma cell marker Multiple Myeloma Oncogene 1 (MUM1) was not expressed. Therefore, the diagnosis was confirmed to be B-cell CLL., Conclusion: Immunophenotyping can help subclassify the type of lymphoid leukemia; however, as tumor cells can show aberrant expression or loss of the CD21 marker, combining immunophenotyping with the PARR assay could yield a more accurate diagnosis., (© 2024. The Author(s).)

Background: The accuracy of blood cell ratios (BCRs) as cost-effective and easily accessible diagnostic and prognostic markers of inflammatory conditions has been investigated in veterinary medicine in recent years., Methods: Neutrophil-to-lymphocyte (NLR), monocyte-to-lymphocyte (MLR), and platelet-to-lymphocyte (PLR) ratios were studied in 195 dogs clinically evaluated and tested for anti-Leishmania infantum (Li) antibodies (Li-seronegative (Li - ), n = 10; Li-seropositive clinically healthy (Li + healthy ), n = 100; Li-seropositive with clinical and/or clinicopathological abnormalities (Li + sick ), n = 85). The Li + sick dogs were classified in LeishVet stages IIa/IIb (Li + IIa/IIb ) (n = 66) and III/IV (Li + III/IV ) (n = 19). BCR relationships with LeishVet clinical stage, antibody levels, and serum protein electrophoretic fraction concentrations were investigated., Results: Higher NLR values were found in Li + , Li + healthy , and Li + IIa/IIb sick dogs compared to Li - dogs (P < 0.001). Higher NLR and MLR were found in Li + sick (NLR, P < 0.001; MLR, P = 0.034) and Li + III/IV dogs (NLR, P < 0.001; MLR, P = 0.005) compared to Li - dogs, and in Li + III/IV dogs (NLR, P = 0.002; MLR, P < 0.001) compared to Li + healthy . All three BCRs were higher in Li + sick (NLR, MLR, P < 0.001; PLR, P = 0.023) and Li + IIa/IIb dogs (NLR P < 0.001; MLR P = 0.001; PLR, P = 0.012) compared to Li + healthy dogs. The BCRs failed to distinguish dogs with moderate (Li + IIa/IIb ) and severe or very severe disease (Li + III/IV ). BCRs demonstrated weak positive correlations with serum globulin fractions and antibody levels, and weak negative correlations with serum albumin level were found. Li + sick dogs presenting hypoalbuminemia showed higher MLR ratios (P = 0.001) than those with normal albumin values., Conclusions: This study shows that BCR measures provide useful information for differentiating antibody-positive healthy and sick dogs at diagnosis. Dogs with hypoalbuminemia showed higher MLR values despite monocytosis being very rare., (© 2024. The Author(s).)

Canine parvovirus (CPV) is a significant threat to canines and is widely distributed worldwide. While vaccination is currently the most effective preventive measure, existing vaccines are not able to offer comprehensive and dependable protection against CPV infection. Hence, there is a need to explore alternative or complementary strategies to tackle this problem. In this study, we present an approach for the efficient screening of canine antibodies targeting CPV using a single B cell antibody technique. We sorted single IgM - IgG + CPV + B cells from canine peripheral blood mononuclear cells using fluorescence-activated cell sorting (FACS) and obtained the variable region genes of heavy and light chains (VH and VL) by nested PCR amplification. Canine monoclonal antibodies were expressed in HEK293 cells, and a total of 60 antibodies were obtained, five of which demonstrated neutralizing activity against CPV. Those findings demonstrate the effectiveness of the method for obtaining canine monoclonal antibodies, which in turn aids in the identification and screening of neutralizing antibodies against various canine pathogens., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

In canine leishmaniosis endemic areas, Leishmania infantum may occur in sympatry with the non-pathogenic Leishmania tarentolae, which is associated to reptiles. The potential infectivity of L. tarentolae for mammals raises questions about the interactions between the two Leishmania species, and the potential cross-immune protection in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in dogs, determining: i) the anti-L. tarentolae antibody production, ii) the duration of the immunity and cytokine expression, and iii) the possible pathogenic effect in the canine host. Twelve purpose-bred beagle dogs were randomly allocated to three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). G1 and G2 dogs were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae strain (RTAR/IT/21/RI-325) isolated from a Tarentola mauritanica gecko. The animals were followed until day 206. Blood, serum, conjunctival swabs and lymph node aspirate samples were collected monthly and bone marrow, liver and spleen biopsies on day 91. Hematological and biochemical parameters were assessed monthly, as well as serology (IFAT and ELISA) and molecular identification of L. tarentolae. Mononuclear cells (PBMC) were obtained to assess the cytokine expression through in vitro stimulation or (re-) infection. Data from this study demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype after derived macrophages (re-) infection, as demonstrated by the expression of IFN-gamma. Therefore, L. tarentolae demonstrated a great potential as a surrogate pathogen and/or immune-prophylaxis/immune-therapy against Leishmania infections in dogs and humans., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mendoza-Roldan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Animal rabies is a potentially fatal infectious disease in mammals, especially dogs. Currently, the number of rabies cases in pet dogs is increasing in several regions of Thailand. However, no passive postexposure prophylaxis (PEP) has been developed to combat rabies infection in animals. As monoclonal antibodies (MAbs) are promising biological therapies for postinfection, we developed a canine-neutralizing MAb against rabies virus (RABV) via the single-chain variable fragment (scFv) platform. Immunized phage-displaying scFv libraries were constructed from PBMCs via the pComb3XSS system. Diverse canine VHVLκ and VHVLλ libraries containing 2.4 × 10 8 and 1.3 × 10 6 clones, respectively, were constructed. Five unique clones that show binding affinity with the RABV glycoprotein were then selected, of which K9RABVscFv1 and K9RABVscFv16 showed rapid fluorescent foci inhibition test (RFFIT) neutralizing titers above the human protective level of 0.5 IU/ml. Finally, in silico docking predictions revealed that the residues on the CDRs of these neutralizing clones interact mainly with similar antigenic sites II and III on the RABV glycoprotein. These candidates may be used to develop complete anti-RABV MAbs as a novel PEP protocol in pet dogs and other animals., (© 2024. The Author(s).)

Interleukin 27 (IL-27) is a cytokine that regulates susceptibility to Leishmania infantum infection in humans and experimental models. This cytokine has not yet been described in canine leishmaniasis (CanL). Therefore, we investigated whether IL-27 has a regulatory role in CanL. The EBI3 and p28 subunits of IL-27 were measured in splenic leukocytes culture supernatant from dogs with CanL and compared to control dogs. We also correlated EBI3 and p28 levels with IL-21, anti-L. infantum antibodies and parasite loads. We performed functional assays followed by IL-27 blockade and measured parasite loads, production of cytokines in splenic leukocytes culture supernatant, and the expression of PD-1, CTLA-4, phospho-Stat-1/3, T-bet, GATA3 and nitric oxide production (NO). Both IL-27 subunits increased in the supernatant of dogs with CanL compared to control dogs. EBI3 and p28 levels showed a moderate positive correlation with IL-21 (r = 0.67, p < 0.0001 and r = 0.45, p < 0.012, respectively), and the EBI3 subunit was positively associated with anti-L. infantum IgG antibodies (r = 0.38, p < 0.040) and parasite load (r = 0.47, p < 0.009). IL-27 and IL-21 participate of immune responses in CanL. IL-27 may be associated with the failure of immunity to control parasite replication via upregulation of the expression of PD-1, CTLA-4, T-bet and NO in splenic leukocytes from dogs with CanL. These findings suggest that the pathways regulated by IL-27 are involved in CanL pathogenesis in the host, and may be targets for new therapies., (© 2024 John Wiley & Sons Ltd.)

Leishmania spp. are an intracellular protozoa present in many countries around the world. In Europe, both the parasite and the disease it causes, leishmaniasis, are endemic in the Mediterranean basin. Clinical signs and severity of disease are highly variable depending on the host in both humans and dogs, traditionally considered the main reservoir of the parasite. The reason for these differences is not known, but it has been speculated that some hosts present immune response, related to activation of Th1 and Th17, capable of controlling the spread of the parasite, and that these immune responses are related to the genetic background of the host. The Ibizan hound, an autochthonous canine breed of the Mediterranean basin, has been postulated as a breed resistant to infection, but other canine breeds evolutionarily close to it and native to this region have not been studied. One of them is the Cirneco dell'Etna, native to the island of Sicily in southern Italy. In this study, the immune response against L. infantum infection in this canine breed was analysed. The results showed that infected dogs of this breed present high levels of several cytokines related to Th1 and Th17 immune response, and significant correlation between serum levels of cytokines related to disease resistance. Further studies are necessary in this canine breed to determine the mechanisms of immune response and genetic background related to L. infantum infection control., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024. Published by Elsevier Ltd.)

Companion dogs have served an important role in cancer immunotherapy research. Sharing similar environments and diets with humans, dogs naturally develop many of the same cancers. These shared exposures, coupled with dogs' diverse genetic makeup, make them ideal subjects for studying cancer therapies. Tumors like osteosarcoma, hemangiosarcoma, soft-tissue sarcoma, and non-Hodgkin lymphoma occur with greater frequency than their counterpart disease in humans. Canine brain tumors allow the study of therapy strategies with imaging, surgery, and radiotherapy equipment in veterinary patients with near-human geometry. Nonspecific immunostimulants, autologous and allogeneic vaccines, immune checkpoint inhibitors, and cellular therapies used in treating canine cancers have been tested in veterinary clinical trials. These treatments have not only improved outcomes for dogs but have also provided valuable insights for human cancer treatment. Advancements in radiation technology and the development of tools to characterize canine immune responses have further facilitated the ability to translate veterinary clinical trial results to human applications. Advancements in immunotherapy of canine tumors have directly supported translation to human clinical trials leading to approved therapies for patients with cancer around the world. The study of immunotherapy in dogs has been and will continue to be a promising avenue for advancing human cancer treatment., (©2024 American Association for Cancer Research.)

Inhalation allergies caused by cats and dogs can lead to a range of discomforting symptoms, such as rhinitis and asthma, in humans. With the increasing popularity of and care provided to these companion animals, the allergens they produce pose a growing threat to susceptible patients' health. Allergens from cats and dogs have emerged as significant risk factors for triggering asthma and allergic rhinitis worldwide; however, there remains a lack of systematic measures aimed at assisting individuals in recognizing and preventing allergies caused by these animals. This review provides comprehensive insights into the classification of cat and dog allergens, along with their pathogenic mechanisms. This study also discusses implementation strategies for prevention and control measures, including physical methods, gene-editing technology, and immunological approaches, as well as potential strategies for enhancing allergen immunotherapy combined with immunoinformatics. Finally, it presents future prospects for the prevention and treatment of human allergies caused by cats and dogs. This review will improve knowledge regarding allergies to cats and dogs while providing insights into potential targets for the development of next-generation treatments.

Background: We aimed to investigate the association between selected inflammatory and immune variables and survival of dogs with myxomatous mitral valve disease (MMVD). We evaluated data of 62 client-owned dogs with MMVD, grouped into preclinical, stable congestive heart failure (CHF) and unstable CHF. Univariate Cox proportional hazards regression analysis was used to quantify the association of white blood cell count, concentrations and percentages of T lymphocytes and their subtypes (T helper lymphocytes, cytotoxic T lymphocytes, double positive T lymphocytes, double negative T lymphocytes) and B lymphocytes with survival. P values < 0.1 in individual groups and P values < 0.05 in the group of all patients were considered significant. Spearman correlation coefficients between significant covariates were calculated to assess the relationships among variables and with survival., Results: In the preclinical group, percentage of double positive T lymphocytes was negatively associated with survival (hazard ratio (HR) = 2.328; P = 0.051). In the unstable CHF, T lymphocyte (HR = 1.613; P = 0.085), cytotoxic T lymphocyte (HR = 1.562; P = 0.048), double positive (HR = 1.751; P = 0.042), and double negative T lymphocyte (HR = 1.613; P = 0.096) concentrations were negatively associated with survival, as well as cytotoxic T lymphocyte (HR = 1.502; P = 0.007) concentration in the group of all patients. The percentage of T helper lymphocytes was positively associated with survival in the unstable CHF (HR = 0.604; P = 0.053) and in the group of all patients (HR = 0.733; P = 0.044). The concentration of cytotoxic T lymphocytes positively correlated with left atrial to aortic ratio (LA/Ao) (rho = 0.259, P = 0.037), and peak velocity of early diastolic mitral flow (rho = 0.259, P = 0.039), whereas the percentage of T helper lymphocytes negatively correlated with left atrial to aortic ratio (LA/Ao) (rho = -0.212, P = 0.090) and early to late mitral flow ratio (rho = -0.232, P = 0.072)., Conclusions: Cytotoxic T lymphocytes, T helper lymphocytes, double positive and double negative T lymphocytes as well as biomarkers cardiac troponin I, N-terminal pro-B-type natriuretic peptide, C-reactive protein are implicated in the progression of MMVD., (© 2024. The Author(s).)

Immune checkpoint inhibitor therapy can provide significant clinical benefit in patients with certain cancer types including melanoma; however, objective responses are only observed for a subset of patients. Mucosal melanoma is a rare melanoma subtype associated with a poor prognosis and, compared with cutaneous melanoma, is significantly less responsive to immune checkpoint inhibitors. Spontaneous canine tumours have emerged as valuable models to inform human cancer studies. In contrast to human melanoma, most canine melanomas are mucosal-an incidence that may be leveraged to better understand the subtype in humans. However, a more comprehensive understanding of the immune landscape of the canine disease is required. Here, we quantify tumour infiltrative T and myeloid cells in canine mucosal (n = 13) and cutaneous (n = 5) melanomas using immunohistochemical analysis of CD3 and MAC387 expression, respectively. Gene expression analysis using the Canine IO NanoString panel was also performed to identify genes and pathways associated with immune cell infiltration. T and myeloid cell densities were variable with geometric means of 158.7 cells/mm 2 and 166.7 cells/mm 2 , respectively. Elevated T cell infiltration was associated with increased expression of cytolytic genes as well as genes encoding the coinhibitory checkpoint molecules PD-1, CTLA-4, TIM-3 and TIGIT; whereas increased myeloid cell infiltration was associated with elevated expression of protumourigenic cytokines. These data provide a basic characterization of the tumour microenvironment of canine malignant melanoma and suggest that, like human melanoma, inherent variability in anti-tumour T cell responses exists and that a subset of canine melanomas may respond better to immunomodulation., (© 2024 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Canine parvovirus (CPV-2) is a highly contagious virus affecting dogs worldwide, posing a significant threat. The VP2 protein stands out as the predominant and highly immunogenic structural component of CPV-2. Soon after its emergence, CPV-2 was replaced by variants known as CPV-2a, 2b and 2c, marked by changes in amino acid residue 426 of VP2. Additional amino acid alterations have been identified within VP2, with certain modifications serving as signatures of emerging variants. In Brazil, CPV-2 outbreaks persist with diverse VP2 profiles. Vaccination is the main preventive measure against the virus. However, the emergence of substitutions presents challenges to conventional vaccine methods. Commercial vaccines are formulated with strains that usually do not match those currently circulating in the field. To address this, the study aimed to investigate CPV-2 variants in Brazil, predict epitopes, and design an in silico vaccine tailored to local variants employing reverse vaccinology. The methodology involved data collection, genetic sequence analysis, and amino acid comparison between field strains and vaccines, followed by the prediction of B and T cell epitope regions. The predicted epitopes were evaluated for antigenicity, allergenicity and toxicity. The final vaccine construct consisted of selected epitopes linked to an adjuvant and optimized for expression in Escherichia coli. Structural predictions confirmed the stability and antigenicity of the vaccine, while molecular docking demonstrated interaction with the canine toll-like receptor 4. Molecular dynamics simulations indicated a stable complex formation. In silico immune simulations demonstrated a progressive immune response post-vaccination, including increased antibody production and T-helper cell activity. The multi-epitope vaccine design targeted prevalent CPV-2 variants in Brazil and potentially other regions globally. However, experimental validation is essential to confirm our in silico findings., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Dogs are important reservoir hosts for Leishmania infantum, the causative agent of visceral leishmaniasis. The complement system, as part of the innate immune defense, is responsible for initiating the fight against pathogens that may invade an organism. A failure of the complement to combat L. infantum may explain, at least in part, why a mammal species is more or less susceptible to visceral leishmaniasis. The objective of this study was to compare the effectiveness of human and dog complement systems against L. infantum parasites. The results showed that dog serum was less effective than human serum at killing promastigote and amastigote-like forms. We also compared the efficiency of human and canine sera in classic and alternative hemolytic assays, as well as the serum efficiency of non-infected and Leishmania-infected dogs. Serum from dogs was less hemolytic than human serum in both pathways tested, but the efficiency of serum from infected dogs was higher than that of non-infected dogs. When testing C3b deposition assays on parasite surfaces, serum from infected dogs was more effective against amastigote-like forms than serum from non-infected dogs. However, both types of serum proved equally effective on promastigotes, while serum from infected dogs was more effective on amastigote-like forms. Considering the efficiency of the complement system, our results indicate that dogs are more susceptible to visceral leishmaniasis than humans are., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Nelder Figueiredo Gontijo reports financial support was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq. Nelder Figueiredo Gontijo reports financial support was provided by Fundação de Amparo à Pesquisa de Minas Gerais - Fapemig. There is no other relationship or activity that may be interpreted as a conflict of interest by the reader. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leptospirosis is a globally distributed zoonosis with multisystemic involvement in canine species, capable of causing a pulmonary hemorrhagic syndrome (LPHS) in the most severe cases. In humans, the neutrophil to lymphocyte ratio (NLR), platelets to lymphocytes (PLR) and systemic immune-inflammation index (SII) have been described as predictors of morbidity and mortality in various pathologies, but no such studies have been developed for canine leptospirosis. Hence, we aimed to assess the usefulness of NLR, PLR and SII in dogs affected with leptospirosis, focusing on those that died or survived after hospitalization, whether or not they developed LPHS. The leptospirosis group was composed by 36 dogs while the control group consisted of 32 healthy dogs. The NLR, associated with inflammation, demonstrated a threefold or greater increase in all leptospirosis groups compared to the control group (median 2.44 ± 1.66) (developing or not LPHS). Dogs that died (median 67.78 ± 158.67), developed LHPS (median 85.17 ± 143.77), or both developed LHPS and died (median 67.78 ± 155,14) had a lower PLR in comparison to the control group (median 101,82 ± 53,75) and the rest of groups, but no statistically significant differences were observed (p > 0.05). The SII was higher in leptospirosis-affected dogs that survived (median 1356,92 ± 2726,29) and statistically significant differences were observed in those who did not develop LPHS (median 1770,41 ± 2630,77; p < 0.05) compared to the control group (median 555,21 ± 313,26). Our data shows that NLR may be used as inflammation indicator, while more studies are needed for PLR and SII in canine leptospirosis., (© 2024. The Author(s).)

Background: Allergen-carrying virus-like particles are effective and safe means of allergen immunotherapy (AIT) in rodent models., Objective: To study the development of allergen-blocking immunoglobulin (Ig)G in dogs injected with Der f 2-carrying enveloped plant-based bioparticles (eBPs)., Materials and Methods: Laboratory beagle dogs were injected intradermally (ID) or subcutaneously (SC) with Der f 2-eBP three times at 2-week intervals. A basophil mediator release assay was used to compare the reactivity of Der f 2-eBPs to that of recombinant Der f 2. Allergen-specific IgG serum levels were determined by immunoblotting and ELISA. The allergen-blocking potential of postvaccination IgG was assessed by Pet Allergy Xplorer (PAX) macroarray and basophil mediator release inhibition assays., Results: The amount of Der f 2 eBPs needed to induce basophil activation was 1000-fold higher than that of the soluble natural allergen. In both immunisation groups, eBP injections caused no adverse events and induced Der f 2-specific IgG, first detected on Day (D)14 and peaking on D41. The co-incubation of sera with a Der f 2-IgE-rich canine serum pool resulted in a mean PAX inhibition of 70% (ID) to 80% (SC) on D41. For both groups, the inhibition of basophil mediator release reached 75% on D28 and D41. The percentage inhibition of PAX and mediator release correlated significantly with Der f 2 IgG levels., Conclusion and Clinical Relevance: Intradermal and subcutaneous injections of Der f 2-eBPs were safe and increased Der f 2-specific IgG. The clinical benefit of immunotherapy will be evaluated in future trials enrolling atopic dogs allergic to house dust mites., (© 2024 ESVD and ACVD.)

Background: Immunoglobulin (Ig)E cross-reactivity has been shown between Dermatophagoides farinae (Df; house dust mite) and the nematode Toxocara canis (Tc), yet its allergen basis is unknown., Objectives: To identify the Df allergens IgE-cross-reactive with those of Tc., Animals: Archived sera from 73 dogs with suspected allergy sensitised to Df., Materials and Methods: We performed a combination of Pet Allergy Xplorer (PAX) and enzyme-linked immunosorbent assay (ELISA) inhibitions with excretory-secretory and somatic (i.e. nematode body) extracts of Tc or recombinant Tc tropomyosin on coats of Df, Der f 15 and Zen-1 (ELISA) or PAX allergens., Results: The ELISA and PAX inhibitions established that there is mutual yet variable cross-reactivity between the Tc excretory-secretory extract, purified Der f 15 and purified Zen-1. This cross-reactivity is likely to involve cross-reactive glycans, as there is no inhibition between the Tc excretory-secretory extract and recombinant Der f 15 without its predicted natural O-glycans. We also confirmed a heterogeneous cross-reactivity between the somatic Tc extract and Der p 11 (paramyosin), as well as between the recombinant Toxo c 3 and Der p 10 tropomyosins. The cross-reactivity among tropomyosins and paramyosins is likely to involve peptidic epitopes, as these recombinant allergens are not glycosylated., Conclusions and Clinical Relevance: In dogs with suspected allergies, the cross-reactivity between Tc and Df for dogs is complex and heterogeneous. Some of the cross-reactive IgE recognises shared glycans on Der f 15 and Zen-1, while some targets peptidic epitopes on shared paramyosins and tropomyosins. We do not exclude that additional cross-reactive allergens between Df and Tc also might exist., (© 2024 ESVD and ACVD.)

Canine osteosarcoma (OSA) is a malignancy that has been shown to modulate the host immune system. Macrophage colony-stimulating factor (M-CSF; CSF1) and interleukin-34 (IL-34; IL34) are both ligands of colony stimulating factor 1 receptor (CSF-1R), and may play a role in the pathogenesis of a variety of human cancers, including OSA. This study aimed to, (1) assess M-CSF and IL-34 expression in canine OSA cell lines and tissue samples, and (2) determine any correlations between M-CSF and IL-34 expression and immune cell infiltrates within canine OSA tissues. Four canine OSA cell lines and canine osteoblasts were treated with control media, TNFα (10 ng/mL) or IL-1β (10 ng/mL) and analysed with RT-qPCR and ELISA. IL-34 and M-CSF mRNA and protein were detectable in all cell lines, however upregulation following TNFα or IL-1β exposure was only consistently observed for transcript expression. Baseline expression of CSF1 and IL34 mRNA in OSA cell lines was equal to or higher than that of canine osteoblasts. All 10 OSA tissue samples expressed IL34 and CSF1 transcripts to varying degrees. Furthermore, CSF1 and IL34 expression both showed a moderate to high degree of correlation with M1 macrophage lineage-associated transcripts (CD80 and IL15RA). There was a moderate degree of correlation between CSF1 and CD163, but no correlation between IL34 and either M2 macrophage-associated transcripts (CD163 and CCL24). In summary, IL-34 and M-CSF are expressed in canine OSA cell lines and tissues, and expression positively correlates with a wide range of immune-related transcripts., (© 2024 John Wiley & Sons Ltd.)

A comprehensive understanding of the tumour immune microenvironment (TIME) is essential for advancing precision medicine and identifying potential therapeutic targets. This study focused on canine urothelial carcinoma (cUC) recognised for its high sensitivity to cyclooxygenase (COX) inhibitors. Using immunohistochemical techniques, we quantified the infiltration of seven immune cell populations within cUC tumour tissue to identify clinicopathological features that characterise the TIME in cUC. Our results revealed several notable factors, including the significantly higher levels of CD3 + T cells and CD8 + T cells within tumour cell nests in cases treated with preoperative COX inhibitors compared to untreated cases. Based on the immunohistochemistry data, we further performed a comparative analysis using publicly available RNA-seq data from untreated cUC tissues (n = 29) and normal bladder tissues (n = 4) to explore the link between COX-prostanoid pathways and the immune response to tumours. We observed increased expression of COX-2, microsomal prostaglandin E2 synthase-1 (mPGES-1) and mPGES-2 in cUC tissues. However, only mPGES-2 showed a negative correlation with the cytotoxic T-cell (CTL)-related genes CD8A and granzyme B (GZMB). In addition, a broader analysis of solid tumours using The Cancer Genome Atlas (TCGA) database revealed similar patterns in several human tumours, suggesting a common mechanism in dogs and humans. Our results suggest that the COX-2/mPGES-2 pathway may act as a cross-species tumour-intrinsic factor that weakens anti-tumour immunity, and that COX inhibitors may convert TIME from a 'cold tumour' to a 'hot tumour' state by counteracting COX/mPGES-2-mediated immunosuppression., (© 2024 The Author(s). Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Transforming growth factor-β (TGFβ) and FoxP3 regulatory T cells (Treg) are involved in human breast carcinogenesis. This topic is not well documented in canine mammary tumors (CMT). In this work, the tumoral TGFβ expression was assessed by immunohistochemistry in 67 malignant CMT and its correlation to previously determined FoxP3, VEGF, and CD31 markers and other clinicopathologic parameters was evaluated. The high levels of TGFβ were statistically significantly associated with skin ulceration, tumor necrosis, high histological grade of malignancy (HGM), presence of neoplastic intravascular emboli and presence of lymph node metastases. The observed levels of TGFβ were positively correlated with intratumoral FoxP3 (strong correlation), VEGF (weak correlation) and CD31 (moderate correlation). Tumors that presented a concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31 markers were statistically significantly associated with parameters of tumor malignancy (high HGM, presence of vascular emboli and nodal metastasis). Additionally, shorter overall survival (OS) time was statistically significantly associated with tumors with an abundant TGFβ expression and with concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31. The presence of lymph node metastasis increased 11 times the risk of disease-related death, arising as an independent predictor of poor prognosis in the multivariable analysis. In conclusion, TGFβ and Treg cells seem involved in tumor progression emerging as potential therapeutic targets for future immunotherapy studies.

Surveillance and sustained control of visceral leishmaniasis (VL) require reliable serodiagnostic tools. rK39, the gold standard antigen for VL diagnosis, is limited by its documented poor sensitivity in certain endemic regions, such as East Africa, and by the longevity of its antibodies, making it difficult to distinguish active from cured infections. In a recent publication in mBio , Roberts et al. (A. J. Roberts, H.B. Ong, S. Clare, C. Brandt, et al., mBio 15:e00859-24, 2024, https://doi.org/10.1128/mbio.00859-24) identified new immunogenic Leishmania candidates in dogs and humans. In dogs, combined antigens LdBPK&#95;290790.1 + LdBPK&#95;362700.1 (D4 +D46) distinguished symptomatic from asymptomatic infections. For humans, LdBPK&#95;323600.1 (D36) antigen produced short-lived antibodies and performed well in patient cohorts from Bangladesh and Ethiopia, but not Kenya. This study adds promising new candidates to our serodiagnostic toolbox but highlights the need for more antigen discovery studies that may have to be focused on regional performance., Competing Interests: The authors declare no conflict of interest.

This study examines household pets as potential epidemiological links between environments contaminated with pathogenic leptospires and humans in Santa Fe, Argentina. The aims of our study were: (a) to characterize the habits and exposure to environmental sources of leptospirosis in the population of dogs and cats attending to municipal spay and neutering campaigns in Santa Fe, Argentina, (b) to assess the seroprevalence of anti-Leptospira antibodies in asymptomatic dogs and cats, (c) to evaluate factors that could increase seropositivity, and (d) to identify spatial clusters of seropositive dogs and cats in the capital city of Santa Fe. From May to November 2022, a cross-sectional serosurvey was conducted during municipal spaying/neutering campaigns. Eligible household dogs and cats were over 6 months old, apparently healthy, and not vaccinated against leptospirosis in the past 6 months. We used microagglutination test (MAT) to assess anti-Leptospira antibodies using a panel of 10 reference strains. We used generalized linear mixed effects models (GLMM) to examine individual and census tract-level risk factors for seropositivity, and local Moran's I statistic for spatial clusters. Results showed higher leptospiral antibody prevalence in dogs (18.2 %) than cats (3.6 %, p = 0.002). Dogs with street access had higher likelihood of being seropositive (OR: 3.8, 95 % CI: 1.2; 11.9), and areas with chronic poverty showed an elevated risk of presenting seropositive animals (RR: 4.0, 95 % CI: 1.1; 14.4). Spatial analysis didn't reveal significant seropositivity clusters among census tracts. These findings shed light on widespread Leptospira seropositivity in pets in this endemic region. Understanding seroprevalence and risk factors can guide public and veterinary health strategies, emphasizing increased leptospirosis vaccination for dogs in vulnerable areas and promoting responsible pet care., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Morphological and immunohistochemical studies of solid arrangement canine mammary carcinomas have shown that the different histological types may be characterized by proliferation of epithelial and/or myoepithelial cells. However, little is known about immunophenotypes and the importance of inflammation as prognostic factors in these neoplasms. The objective of the present study was to characterize the immunophenotype and degree of inflammation in the solid type of canine mammary neoplasm and to investigate their association with metastasis, Ki-67 index, tumour size, necrosis and survival. Sixty-five carcinomas with solid pattern, basaloid carcinomas, solid papillary carcinomas, malignant adenomyoepitheliomas (MAMEs) or malignant myoepitheliomas (MMEs) were investigated. Luminal A, luminal B HER2 negative and HER2 positive, HER2 overexpressed and triple negative immunophenotypes were immunolabelled as were Ki-67 protein and cyclooxygenase-2 (Cox-2). Histological peritumoural and intratumoural inflammatory infiltrates were graded (distribution × intensity) and the presence of necrosis identified. We found a statistical difference between histological types and immunophenotypes, with MME and MAME having a higher occurrence of luminal A, whereas most neoplasms had the luminal B HER-negative immunophenotype. There was no correlation between immunophenotype and degree of peri- and intratumoural inflammation, nodal metastasis, necrosis or tumour size. An increased degree of peri- and intratumoural inflammation was significantly associated with lymph node metastasis, and more severe intratumoural inflammation was associated with the presence of tumour necrosis. Tumour size, Ki-67 index and Cox-2 score were not associated with inflammation in either peri- or intratumoural regions. No difference was observed in survival in relation to immunophenotype or degree of inflammation, but the Cox regression model revealed that nodal metastasis influenced the risk of death., (Copyright © 2024. Published by Elsevier Ltd.)

Immunotherapy is a promising alternative treatment for canine mast cell tumour (MCT). However, evasion of immune recognition by downregulating major histocompatibility complex (MHC) molecules might decline treatment efficiency. Enhancing MHC expression through interferon-gamma (IFN-γ) is crucial for effective immunotherapy. In-house and reference canine MCT cell lines derived from different tissue origins were used. The impacts of IFN-γ treatment on cell viability, expression levels of MHC molecules, as well as cell apoptosis were evaluated through the MTT assay, RT-qPCR and flow cytometry. The results revealed that IFN-γ treatment significantly influenced the viability of canine MCT cell lines, with varying responses observed among different cell lines. Notably, IFN-γ treatment increased the expression of MHC I and MHC II, potentially enhancing immune recognition and MCT cell clearance. Flow cytometry analysis in PBMCs-mediated cytotoxicity assays showed no significant differences in overall apoptosis between IFN-γ treated and untreated canine MCT cell lines across various target-to-effector ratios. However, a trend towards higher percentages of late and total apoptotic cells was observed in the IFN-γ treated C18 and CMMC cell lines, but not in the VIMC and CoMS cell lines. These results indicate a variable response to IFN-γ treatment among different canine MCT cell lines. In summary, our study suggests IFN-γ's potential therapeutic role in enhancing immune recognition and clearance of MCT cells by upregulating MHC expression and possibly promoting apoptosis, despite variable responses across different cell lines. Further investigations are necessary to elucidate the underlying mechanisms and evaluate IFN-γ's efficacy in in vivo models., (© 2024. The Author(s).)

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral-immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral-immunocytokine at 12 mg/m 2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral-immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Immunomodulatory antibody drugs that modulate the function of immune checkpoint molecules, such as programmed death receptor-1 (PD-1) and programmed cell death ligand 1 (PD-L1), have been established as new cancer treatments in human medicine. In recent years, there have also been reports on antibodies that inhibit immune checkpoint molecules in dogs, and clinical trials using such antibodies for canine cancer have been gradually increasing in number. Because inhibitory antibodies restore T-cell function by inhibiting the binding of PD-1 on T cells and its ligand PD-L1, the quality of antibody function has been evaluated using activated T cells or peripheral blood mononuclear cells isolated from healthy dogs; however, the assays and dogs used significantly vary. Therefore, in the present study, we developed a reporter gene assay using reporter cells (Jurkat/NFATluc/cPD1) and effector cells (CTAC/OKT3/cPDL1). Jurkat/NFATluc/cPD1 were generated by introducing both of the NFAT-responsive luciferase gene as a marker of T-cell signaling and canine PD-1, into a human T lymphoid cell line, Jurkat. CTAC/OKT3/cPDL1 were generated by introducing single-chain FV (scFV) of anti-human CD3 antibody (OKT3) and canine PD-L1 into a canine thyroid carcinoma cell line, CTAC. Ligation of PD-1 on Jurkat/NFATluc/cPD1 via binding of PD-L1 on CTAC/OKT3/cPDL1 suppressed NFAT luciferase activity induced by CD3 ligation by scFV of OKT3. The addition of anti-canine PD-1 and PD-L1 antibodies, both of which were previously developed in our laboratory, restored this suppression with high sensitivity, although the anti-human PD-L1 antibody atezolizumab induced a very weak restoration. This assay is an useful method for functionally evaluating the inhibition of canine PD-1 and PD-L1 binding., Competing Interests: Declaration of Competing Interest T.M. received research funding from Nippon Zenyaku Kogyo Co., Ltd. The rest of the authors declare no other potential conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pemphigus foliaceus (PF) is an autoimmune skin disease of dogs characterized by intraepidermal pustules containing neutrophils and dissociated keratinocytes that develop in association with circulating and tissue-bound IgG autoantibodies. A subset of IgG autoantibodies in canine PF target desmocollin-1 (DSC1), a component of intercellular adhesion complexes within the epidermis. Passive transfer of IgG autoantibodies from canine PF sera to mice was previously shown to induce skin disease in the absence of infiltrating neutrophils. In attempts to identify a mechanism responsible for neutrophil recruitment, past studies evaluated the prevalence of IgA autoantibodies in canine PF sera where they were found in <20% of affected dogs. We re-evaluated the prevalence of anti-DSC1 IgA in canine PF due to concerns regarding the sensitivity of previously used methods. We hypothesized that anti-DSC1 IgA are present in most dogs with PF but have been under-detected due to competition with concurrent anti-DSC1 IgG for binding to their mutual antigenic target. Despite removing approximately 80% of IgG from patient sera using affinity chromatography, we did not detect an increase in anti-DSC1 IgA by performing indirect immunofluorescence on canine DSC1-transfected HEK293T cells. Taken together, our results do not support a role for pathogenic IgA in canine PF., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tyler J.M. Jordan reports financial support was provided by Morris Animal Foundation. Tyler J.M. Jordan reports financial support was provided by National Institutes of Health. Zhi Liu reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background/aim: The activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been implicated in canine soft tissue sarcoma (STS) and may serve as a prognostic marker. This study investigated the correlation between PI3K/Akt activation in tumor cells and tumor-infiltrating lymphocytes (TILs)., Materials and Methods: A total of 59 STS samples were labeled via immunohistochemistry to calculate the density of TILs, including CD3+ T cells, CD8+ T cells, CD20+ B cells, and FOXP3+ regulatory T cells., Results: Forty-eight samples (81.3%) had intra-tumoral TILs with a high density of CD3+ T cells (mean: 283.3 cells/mm 2 ) and CD8+ T cells (mean: 134.8 cells/mm 2 ). Conversely, CD20+ B cells (mean: 73.6 cells/mm 2 ) and FOXP3+ regulatory T cells (mean: 9.2 cells/mm 2 ) were scarce. The abundance of CD3+/CD8+, CD3+/CD20+, and CD8+/CD20+ TILs were highly correlated in multivariate analyses (r=0.895, 0.946, and 0.856, respectively). Nonetheless, TIL density was unrelated to clinicopathological parameters (sex, age, tumor location, breed) and tumor grade. The abundance of CD8+ T cells was positively correlated with the activation of PI3K/Akt, indicating that samples with high levels of phospho-Akt and phospho-S6 tend to have a higher CD8+ T cell density (p=0.0032 and 0.0218, respectively). Furthermore, TIL density was correlated with the Ki-67 index, a tumor proliferation and growth marker. Samples with a high Ki-67 index had a significantly higher abundance of CD3+ T cells, CD8+ T cells, and CD20+ B cells (p=0.0392, 0.0254, 0.0380, respectively)., Conclusion: PI3K/Akt pathway activation may influence the infiltration of CD8+ T cells within the tumor microenvironment in canine STS. Prospective studies involving a higher number of cases are warranted to confirm these findings., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Little research is available on acquired immunity to rabies in dogs and cats from Central Africa, particularly regarding the legal movements of pets. Movement of domestic animals from rabies-endemic countries like Cameroon to rabies free areas poses one of the main risks for rabies introduction into rabies-free areas. Thus, the aim of this study was to assess the effect of various risk factors on rabies vaccine efficacy in Cameroonian. Since the dependent variable, rabies neutralizing titres, were censored from above (right-censoring), Generalized Additive Model for Location, Scale and Shape (GAMLSS) was used in the analysis. Overall, 85.7% of dogs and 100% of cats had titres greater than or equal to 0.5 IU/mL, which is considered protective. Additionally, compared to cats, the value of the rabies-neutralizing serum titres in dogs was on average smaller by 2.3 IU/mL. For each additional year of age, the value of the rabies-neutralizing serum titre, on average, increased by approximately 0.14 IU/mL. Finally, for each 30 additional days between the date of the last rabies vaccination and the date of the sampling, the value the rabies neutralizing titre, on average, decreased by approximately 0.10 IU/mL, given the species and age at sampling were equivalent. These results are useful for assessing risk and improving surveillance to prevent the introduction of rabies into a country via the international movement of animals., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest. The views and opinions expressed in this article are those personal of the authors and do not necessarily reflect the official policy or position of the World Organisation for Animal Health (OIE/WOAH)., (Copyright © 2023. Published by Elsevier Ltd.)

Canine atopic dermatitis (CAD) is a chronic and inflammatory skin condition with a multifaceted origin, involving genetic factors, skin barrier abnormalities, immune responses, and hypersensitivity to various allergens. Interleukin 33 (IL-33), released by keratinocytes upon cellular injury, plays a crucial role in atopic dermatitis pathogenesis by inducing Th2 lymphocyte-mediated immune responses. This study aimed to evaluate IL-33 expression in dogs with atopic dermatitis and compare it to a control group. Forty-nine dogs were included, with 39 having atopic dermatitis, subdivided into groups based on clinical characteristics, and ten in the control group. Lesion and pruritus scores were assessed, and incisional biopsies were analyzed for dermatopathological characteristics. IL-33 expression was evaluated using immunohistochemistry, the analyses were blinded, based on the measurement of immunostaining areas using Image Pro-Plus software, version 4.5, relying on a semi-automatic color segmentation method, where the tissue immunostaining area for each biomarker was artificially delimited and quantified. Statistically significant differences in IL-33 immunostaining were found among groups (P=0.0005). Lichenified dogs (group 4) exhibited higher immunostaining compared to erythema (group 3) (P=0.0006), alesional pruritus (group 2) (P=0.0261), and the control group (group 1) (P=0.0079). IL-33 immunostaining increased with lesion progression, strongly correlating with lesion scores (P<0.0001), particularly in patients with chronic lesions characterized by erythema and lichenification. These findings suggest IL-33's significant role in canine atopic dermatitis pathogenesis and its association with lesion and inflammation scores during the chronic phase. This suggests potential therapeutic interventions targeting IL-33 or its receptors, though further studies are needed to explore these possibilities., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Fernanda Borek reports financial support was provided by Coordination of Higher Education Personnel Improvement. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Pugs commonly present with thoracolumbar myelopathy, also known as pug dog myelopathy (PDM), which is clinically characterised by progressive signs involving the pelvic limbs, no apparent signs of pain and, often, incontinence. In addition to meningeal fibrosis and focal spinal cord destruction, histopathology has confirmed lymphohistiocytic infiltrates in the central nervous system (CNS) in a considerable number of pugs with PDM. Lymphohistiocytic CNS inflammation also characterises necrotising meningoencephalitis (NME) in pugs. This study aimed to investigate the potential contribution of an immunological aetiology to the development of PDM., Methods: The concentrations of glial fibrillary acidic protein (GFAP) in serum and CSF and of anti-GFAP autoantibodies in CSF were measured with an ELISA. In addition, a commercial test was used for genetic characterisation of the dog leukocyte antigen class II haplotype, which is associated with NME susceptibility., Results: This study included 87 dogs: 52 PDM pugs, 14 control pugs, four NME pugs and 17 dogs of breeds other than pugs that were investigated for neurological disease (neuro controls). Anti-GFAP autoantibodies were present in 15 of 19 (79%) of the PDM pugs tested versus six of 16 (38%) of the neuro controls tested (p = 0.018). All 18 PDM pugs evaluated had detectable CSF GFAP. Serum GFAP was detected in two of three (67%) of the NME pugs and in two of 11 (18%) of the control pugs but not in any of the 40 tested PDM pugs. Male pugs heterozygous for the NME risk haplotype had an earlier onset of clinical signs (70 months) compared to male pugs without the risk haplotype (78 months) (p = 0.036)., Limitations: The study was limited by the lack of healthy dogs of breeds other than pugs and the small numbers of control pugs and pugs with NME., Conclusions: The high proportion of PDM pugs with anti-GFAP autoantibodies and high CSF GFAP concentrations provide support for a potential immunological contribution to the development of PDM., (© 2024 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

Chronic inflammatory enteropathy (CIE) is a common condition in dogs causing recurrent or persistent gastrointestinal clinical signs. Pathogenesis is thought to involve intestinal mucosal inflammatory infiltrates, but histopathological evaluation of intestinal biopsies from dogs with CIE fails to guide treatment, inform prognosis, or correlate with clinical remission. We employed single-cell RNA sequencing to catalog and compare the diversity of cells present in duodenal mucosal endoscopic biopsies from 3 healthy dogs and 4 dogs with CIE. Through characterization of 35,668 cells, we identified 31 transcriptomically distinct cell populations, including T cells, epithelial cells, and myeloid cells. Both healthy and CIE samples contributed to each cell population. T cells were broadly subdivided into GZMA high (putatively annotated as tissue resident) and IL7R high (putatively annotated as non-resident) T cell categories, with evidence of a skewed proportion favoring an increase in the relative proportion of IL7R high T cells in CIE dogs. Among the myeloid cells, neutrophils from CIE samples exhibited inflammatory (SOD2 and IL1A) gene expression signatures. Numerous differentially expressed genes were identified in epithelial cells, with gene set enrichment analysis suggesting enterocytes from CIE dogs may be undergoing stress responses and have altered metabolic properties. Overall, this work reveals the previously unappreciated cellular heterogeneity in canine duodenal mucosa and provides new insights into molecular mechanisms which may contribute to intestinal dysfunction in CIE. The cell type gene signatures developed through this study may also be used to better understand the subtleties of canine intestinal physiology in health and disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Manchester, Ammons, Lappin and Dow.)

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system., (© 2024. The Author(s).)

Chimeric antigen receptor (CAR) T-adoptive cell therapy has transformed the treatment of human hematologic malignancies. However, its application for the treatment of solid tumors remains challenging. An exciting avenue for advancing this field lies in the use of pet dogs, in which cancers that recapitulate the biology, immunological features, and clinical course of human malignancies arise spontaneously. Moreover, their large size, outbred genetic background, shared environment with humans, and immunocompetency make dogs ideal for investigating and optimizing CAR therapies before human trials. Here, we will outline how challenges in early clinical trials in patients with canine lymphoma, including issues related to autologous CAR T-cell manufacturing, limited CAR T-cell persistence, and tumor antigen escape, mirrored challenges observed in human CAR T trials. We will then highlight emerging adoptive cell therapy strategies currently under investigation in dogs with hematological and solid cancers, which will provide crucial safety and efficacy data on novel CAR T regimens that can be used to support clinical trials. By drawing from ongoing studies, we will illustrate how canine patients with spontaneous cancer may serve as compelling screening platforms to establish innovative CAR therapy approaches and identify predictive biomarkers of response, with a specific emphasis on solid tumors. With increased funding for canine immunotherapy studies, multi-institutional investigations are poised to generate highly impactful clinical data that should translate into more effective human trials, ultimately benefiting both human and canine cancer patients., (©2024 American Association for Cancer Research.)

The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV., Competing Interests: Declaration of Competing Interest All the authors have no conflicting financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neutrophils, a crucial element of the host defense system, develop extracellular traps against helminth parasites. Neutrophils accumulate around the larvae of Toxocara canis (T. canis) in the tissues of the organism. This study aimed to determine the reaction in canine neutrophils after incubation with infective stage T. canis larvae (L3) in vitro. Most L3 were still active and moved between the extracellular traps (NETs) after 60-min incubation. NETs were not disintegrated by L3 movement. The L3 was only immobilized by NETs, entrapped larvae were still motile between the traps at the 24 h incubation. NETs were observed not only to accumulate around the mouth, excretory pole or anus but also the entire body of live L3. The extracellular DNA amount released from the canine neutrophils after being induced with phorbol 12-myristate 13-acetate was not affected by T. canis excretory/secretory products obtained from 250 L3. To the Authors'knowledge, the extracellular trap structures was firstly observed in canine neutrophils against T. canis L3 in vitro. NETs decorated with myeloperoxidase, neutrophil elastase and histone (H3) were observed under fluorescence microscope. There were not significant differences in the amount of extracellular DNA (P > 0.05), but the morphological structure of NETs was different in the live and head-inactivated T. canis larvae., Competing Interests: Declaration of Competing Interest The authors declare they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Canine Parvovirus type 2 (CPV-2) is a highly contagious virus that can cause severe systemic disease with gastroenteric symptoms in dogs, particularly in young puppies. Originating from the feline parvovirus in the late 1970s, it swiftly propagated globally, instigating a pandemic in dogs. Despite vaccination advancements, CPV-2 remains a substantial challenge for veterinary professionals and pet owners. This study aimed to contribute knowledge about the current situation of CPV-2 among dogs in southern Brazil. In this study, the sera of 125 dogs (mostly with gastroenteritis symptoms) were screened for antibodies against CPV-2 and their faeces for the virus itself. The results showed that 40% (50/125) of dogs were infected with CPV-2. Most animals (65.5%) had previously been exposed to CPV-2 (with serotitres equal or above 1:40), and only 37.6% had protective antibody titres equal or above 1:80. The findings have also demonstrated that vaccination against CPV-2 significantly reduced the risk of infection, with positive cases decreasing from 56.9% (unvaccinated) to 2.0% (fully vaccinated). Furthermore, the prevalence of CPV-2 decreased as dogs aged, with younger dogs and those with an incomplete or non-existent vaccination history at the highest risk of infection. In conclusion, this study provides valuable insight into the prevalence and risk factors associated with CPV-2 infection in dogs in southern Brazil, thereby providing valuable knowledge for the improvement of veterinary care and pet health., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Bordetella bronchiseptica is a pathogen causing respiratory infections in mammals. With the improving understanding of companion animals' welfare, addressing the side effects of bordetella vaccine gains importance in dogs. Studies on diverse subunit vaccines are actively pursued in humans to safely and effectively control bordetellosis. Therefore, our objective was to develop a canine bordetella vaccine inspired by human vaccine development. We evaluated the immunogenicity of the two bacterial components: the outer membrane proteins (OMPs) and the dermonecrotic toxin (DNT) from a canine isolate of B. bronchiseptica. In-silico analysis identified eight domains of DNT, and Domain 3 was selected as the most promising antigen candidate. Additionally, the OMPs were extracted and examined using SDS-PAGE and Western blot analysis. The distinct immunological characteristic of OMPs and DNT-3 were examined individually and in combination. Gene expression and cytokine production were also evaluated in DH82 cells after stimulation with those antigens. Treatment with OMPs resulted in higher level of Th1 related cytokines, while DNT-3 induced a predominant response associated with Th17 and Th2 in the cytokine production. Synergistic effects were observed exclusively on IL-23, indicating increase of a potential risk of side effects when OMPs and DNT act together. These findings provide valuable insights into the reactogenicity of conventional Bordetella vaccines. Further, the presented preclinical data in this study offer an alternative method of the development for an optimal next-generation Bordetella vaccine for companion animals and humans, replacing the acellular vaccines containing both toxin and protein components., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The pathogenesis of anal sacculitis has not been extensively investigated, although atopic dogs seem to be predisposed to the disease. The aim of this study was therefore to characterize and compare the bacterial microbiota and pro-inflammatory cytokines in the anal sacs of dogs from three groups (healthy dogs, untreated atopic dogs and atopic dogs receiving antipruritic treatment or allergen-specific immunotherapy) in order to determine whether changes could be at the origin of anal sacculitis in atopic dogs. Bacterial populations of anal sac secretions from fifteen healthy dogs, fourteen untreated and six treated atopic dogs were characterized by sequencing the V4 region of the 16S rRNA gene using Illumina technology. Proinflammatory cytokines were analyzed with the Luminex multiplex test. Community membership and structure were significantly different between the anal sacs of healthy and untreated atopic dogs (P = 0.002 and P = 0.003, respectively) and between those of untreated and treated atopic dogs (P = 0.012 and P = 0.017, respectively). However, the community structure was similar in healthy and treated atopic dogs (P = 0.332). Among the proinflammatory cytokines assessed, there was no significant difference between groups, except for interleukin 8 which was higher in the anal sacs of untreated atopic dogs compared to treated atopic dogs (P = 0.02), and tumor necrosis factor-alpha which was lower in the anal sacs of healthy dogs compared to treated atopic dogs (P = 0.04). These results reveal a dysbiosis in the anal sacs of atopic dogs, which may partially explain the predisposition of atopic dogs to develop bacterial anal sacculitis. Treatments received by atopic dogs (oclacitinib, desloratadine and allergen-specific immunotherapy) shift the microbiota of the anal sacs towards that of healthy dogs. Further studies are required to identify significant cytokines contributing to anal sacculitis in atopic dogs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 C. Bergeron et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Canine kobuvirus (CaKoV) is a pathogen associated with canine gastrointestinal disease (GID). This study examined 327 rectal swabs (RS), including 113 from Vietnam (46 healthy, 67 with GID) and 214 from Thailand (107 healthy and 107 with GID). CaKoV was detected in both countries, with prevalences of 28.3% (33/113) in Vietnam and 7.9% (17/214) in Thailand. Additionally, CaKoV was found in both dogs with diarrhea and healthy dogs. CaKoV was mainly found in puppies under six months of age (30.8%). Co-detection with other canine viruses were also observed. The complete coding sequence (CDS) of nine Vietnamese and four Thai CaKoV strains were characterized. Phylogenetic analysis revealed a close genetic relationship between Vietnamese and Thai CaKoV strains, which were related to the Chinese strains. CDS analysis indicated a distinct lineage for two Vietnamese CaKoV strains. Selective pressure analysis on the viral capsid (VP1) region showed negative selection, with potential positive selection sites on B-cell epitopes. This study, the first of its kind in Vietnam, provides insights into CaKoV prevalence in dogs of different ages and healthy statuses, updates CaKoV occurrence in Thailand, and sheds light on its molecular characteristics and immune evasion strategies., (© 2024. The Author(s).)

The Nova Scotia Duck Tolling Retriever (NSDTR) is predisposed to immune mediated rheumatic disease (IMRD), steroid-responsive meningitis-arteritis (SRMA) and certain forms of cancer. Cytokines are the main regulators of the immune system. Interleukin 2 is a cytokine involved in activation of T regulatory cells, playing a role in central tolerance and tumor immunity. Interleukin 12 and interleukin 23 share the same subunit, p40, and are both pro-inflammatory cytokines. The aim of this study was to compare levels of IL-2 in healthy NSDTRs to those with cancer or autoimmune disease and to compare levels of IL-12/IL-23p40 in healthy NSDTRs and beagles versus NSDTRs with cancer or autoimmune disease. 62 dogs were included in the analysis of IL-12/IL-23p40; healthy NSDTRs (n = 16), healthy beagles (n = 16), NSDTRs autoimmune (n = 18) and NDSTRs lymphoma/mastocytoma (n = 12) and 68 dogs for IL-2; healthy (n = 20), autoimmune (n = 36) and lymphoma/mastocytoma/adenocarcinoma (n = 12). NSDTRs with autoimmune disease had higher levels of IL-12/IL-23p40 compared to healthy dogs (p = 0.008). NSDTRs with lymphoma also had higher levels of IL-12/IL-23p40 compared to healthy NSDTRs (p = 0.002). There was no difference in levels of IL-2 between healthy and diseased NSDTR. Statistical analysis was performed using Bonferroni corrections for multiple testing. These findings can contribute to the knowledge of autoimmune disease and cancer in dogs., (© 2024. The Author(s).)

Monoclonal antibodies targeting immune checkpoints have revolutionized oncology. Yet, the effectiveness of these treatments varies significantly among patients, and they are associated with unexpected adverse events, including hyperprogression. The murine research model used in drug development fails to recapitulate both the functional human immune system and the population heterogeneity. Hence, a novel model is urgently needed to study the consequences of immune checkpoint blockade. Dogs appear to be uniquely suited for this role. Approximately 1 in 4 companion dogs dies from cancer, yet no antibodies are commercially available for use in veterinary oncology. Here we characterize two novel antibodies that bind canine PD-1 with sub-nanomolar affinity as measured by SPR. Both antibodies block the clinically crucial PD-1/PD-L1 interaction in a competitive ELISA assay. Additionally, the antibodies were tested with a broad range of assays including Western Blot, ELISA, flow cytometry, immunofluorescence and immunohistochemistry. The antibodies appear to bind two distinct epitopes as predicted by molecular modeling and peptide phage display. Our study provides new tools for canine oncology research and a potential veterinary therapeutic., Competing Interests: BV is a consultant for Moravian Biotechnology, who originally produced the PD-1 monoclonal antibodies used in this study. The company did not provide financial support or have any influence over the design, execution, or interpretation of the data. MP is a founder of CanCan Diagnostics, which had no involvement in the study and its results, and declares no conflict of interest. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kocikowski, Dziubek, Węgrzyn, Hrabal, Zavadil-Kokas, Vojtesek, Alfaro, Hupp and Parys.)

Background: Dysregulated T lymphocyte response is thought to play a key role in chronic intestinal inflammation (CIE)., Objectives: To evaluate the presence of changes in peripheral and intestinal T lymphocyte subsets and to describe potential immune and inflammatory biomarkers in dogs with CIE., Animals: Sixteen healthy dogs and 26 dogs were diagnosed with CIE., Methods: Prospective case-control study evaluating peripheral and intestinal T lymphocytes using flow cytometry and inflammatory markers obtained from complete blood cell counts., Results: Dogs with CIE had higher peripheral activated T helper (Th) lymphocytes (87/μL [18-273] CIE, 44/μL [16-162] healthy control (HC, P = .013) and regulatory T cells (Treg; 108/μL [2-257] CIE, 34/μL [1-114] HC, P = .004). In the intestinal epithelium, CIE dogs presented lower percentages of Th (4.55% [1.75-18.67] CIE, 8.77% [3.79-25.03] HC, P = .002), activated Th cells (0.16% [0.02-0.83] CIE, 0.33% [0.05-0.57] HC, P = .03) and CD4/CD8 ratio (0.08 [0.02-0.39] CIE, 0.21 [0.07-0.85] HC, P = .003). Conversely, higher percentage of activated T cytotoxic cells (20.24% [3.12-77.12] CIE, 12.32% [1.21-39.22] HC, P = .04) and interferon-gamma (IFN-γ) producing T lymphocytes (7.36% [0.63-55.83] CIE, 1.44% [0.00-10.56] HC, P = .01) within the epithelium was observed. In the lamina propria the percentage of Treg lymphocytes was higher (6.02% [1.00-21.48] CIE, 3.52% [0.18-10.52] HC, P = .02)., Conclusions and Clinical Importance: Systemic and intestinal immune alterations occur in dogs with CIE suggesting that blood IFN-γ producing T lymphocytes and the systemic immune-inflamation index (SII) could potentially serve as biomarkers for the disease., (© 2024 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Tumor-infiltrating lymphocyte (TIL) density plays an important role in anti-tumor immunity and is associated with patient outcome in various human and canine malignancies. As a first assessment of the immune landscape of the tumor microenvironment in canine renal cell carcinoma (RCC), we retrospectively analyzed clinical data and quantified CD3, FoxP3, and granzyme B immunostaining in formalin-fixed paraffin-embedded tumor samples from 16 dogs diagnosed with renal cell carcinoma treated with ureteronephrectomy. Cell density was low for all markers evaluated. Increased numbers of intratumoral FoxP3 labelled (+) cells, as well as decreased granzyme B+: FoxP3+ TIL ratio, were associated with poor patient outcomes. Our initial study of canine RCC reveals that these tumors are immunologically cold and Tregs may play an important role in immune evasion., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Matthew Atherton reports financial support was provided by National Institutes of Health, NCI, K08CA252619. Charles-Antoine Assenmacher reports financial support was provided by National Institutes of Health Shared Instrumentation Grant (S10 OD023465–01A1). Charles-Antoine Assenmacher reports financial support was provided by Abramson Cancer Center (P30 CA016520). Jennifer Lenz reports financial support was provided by Miso Harris Fund donated to Penn Vet Comprehensive Cancer Care service. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To evaluate the safety and efficacy of human intravenous immunoglobulin in dogs with newly diagnosed malignancy and presumed secondary immune-mediated thrombocytopenia., Materials and Methods: Twelve client-owned dogs with newly diagnosed malignant disease and presumed secondary immune-mediated thrombocytopenia were prospectively enrolled to receive a single infusion of human intravenous immunoglobulin at a dose of 0.5 to 1 mg/kg intravenous over 8 hours. A complete treatment response was defined as a platelet estimation of ≥40,000 platelets/μL within 24 hours and a partial response within 48 hours from the completion of human intravenous immunoglobulin infusion. No treatment response was defined as a platelet estimation remaining <40,000 platelets/μL over 48 hours from the completion of the human intravenous immunoglobulin infusion. This pilot study had a prospective, open-label, uncontrolled design., Results: Out of the 12 enrolled dogs, seven completed the study. A complete treatment response to human intravenous immunoglobulin was identified in one lymphoma dog and a partial response was noted in another lymphoma dog. The remaining 10 dogs had no response to human intravenous immunoglobulin. No clinically relevant adverse reactions to human intravenous immunoglobulin occurred in any of the 12 initially enrolled dogs during the infusion and over a 3-month follow-up period for the seven surviving dogs., Clinical Significance: The results of this study suggest that the use of human intravenous immunoglobulin in dogs with newly diagnosed malignant disease and presumed secondary immune-mediated thrombocytopenia appears safe, but not effective for the treatment of thrombocytopenia. Larger multi-centre, prospective, double-blinded, placebo-controlled, outcome-based, malignancy-specific studies are needed to further evaluate these preliminary findings., (© 2024 British Small Animal Veterinary Association.)

Immuno-oncology research has brought to light the paradoxical role of immune cells in the induction and elimination of cancer. Programmed cell death protein 1 (PD1), expressed by tumor-infiltrating lymphocytes, and programmed cell death ligand 1 (PDL1), expressed by tumor cells, are immune checkpoint proteins that regulate the antitumor adaptive immune response. This study aimed to validate commercially available PDL1 antibodies in canine tissue and then, applying standardized methods and scoring systems used in human pathology, evaluate PDL1 immunopositivity in different types of canine tumors. To demonstrate cross-reactivity, a monoclonal antibody (22C3) and polyclonal antibody (cod. A1645) were tested by western blot. Cross-reactivity in canine tissue cell extracts was observed for both antibodies; however, the polyclonal antibody (cod. A1645) demonstrated higher signal specificity. Canine tumor histotypes were selected based on the human counterparts known to express PDL1. Immunohistochemistry was performed on 168 tumors with the polyclonal anti-PDL1 antibody. Only membranous labeling was considered positive. PDL1 labeling was detected both in neoplastic and infiltrating immune cells. The following tumors were immunopositive: melanomas (17 of 17; 100%), renal cell carcinomas (4 of 17; 24%), squamous cell carcinomas (3 of 17; 18%), lymphomas (2 of 14; 14%), urothelial carcinomas (2 of 18; 11%), pulmonary carcinomas (2 of 20; 10%), and mammary carcinomas (1 of 31; 3%). Gastric (0 of 10; 0%) and intestinal carcinomas (0 of 24; 0%) were negative. The findings of this study suggest that PDL1 is expressed in some canine tumors, with high prevalence in melanomas., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)