Padronização de um teste ELISA indireto para diagnóstico da cinomose canina / Standardization of an indirect ELISA test for canine diagnosis diagnosis

A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.

Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.

Feeding treats containing cannabidiol (CBD) did not alter canine immune response to immunization with a novel antigen.

Due to the potential risk for cannabidiol (CBD) to negatively impact the immune system, the objective of the current study was to evaluate the effect of CBD on the canine immune response to immunization with a novel antigen, keyhole limpet hemocyanin (KLH). Thirty-two dogs (22.4 ± 6.3 kg BW) were utilized in a completely randomized design with treatments consisting of 5 mg CBD/kg BW/d and a control administered orally via treats. After a 7-d acclimation to treatments, dogs were immunized with 10 mg/dog of KLH via intramuscular injection into the semimembranosus muscle region, which was repeated in 14 d. Blood samples were collected at baseline and weekly for 28 d after initial KLH immunization for analysis of hematology, serum chemistry, and immunoglobulins. Data were analyzed using the MIXED procedure in SAS including the fixed effects of treatment, day, and the treatment by day interaction. Both primary and secondary KLH immunization produced robust immune responses. Most hematological and serum chemistry variables remained within normal reference ranges for dogs across both treatments throughout the study. Alkaline phosphatase, while within normal reference range and similar between treatments at baseline and on d 7 (P = 0.994 and 0.183, respectively), was elevated for CBD-treated dogs versus control on d 14, 21, and 28 (P = 0.006, 0.027, and 0.014, respectively). Both total and KLH-specific IgG and IgM were similar between treatments throughout the study (P > 0.05), although total IgM peaked earlier in control dogs compared to those receiving CBD. Despite the minor shift in the timing of the total IgM peak, CBD did not appear to exhibit humoral immunosuppressive effects when supplemented at 5 mg/kg BW/d. However, this work does highlight the potential for CBD to alter liver function and the need for further safety evaluations of CBD use in dogs utilizing longer-term studies and multiple CBD doses.

Development of infrastructure for a systemic multidisciplinary approach to study aging in retired sled dogs.

Canines represent a valuable model for mammalian aging studies as large animals with short lifespans, allowing longitudinal analyses within a reasonable time frame. Moreover, they develop a spectrum of aging-related diseases resembling that of humans, are exposed to similar environments, and have been reasonably well studied in terms of physiology and genetics. To overcome substantial variables that complicate studies of privately-owned household dogs, we have focused on a more uniform population composed of retired Alaskan sled dogs that shared similar lifestyles, including exposure to natural stresses, and are less prone to breed-specific biases than a pure breed population. To reduce variability even further, we have collected a population of 103 retired (8-11 years-old) sled dogs from multiple North American kennels in a specialized research facility named Vaika. Vaika dogs are maintained under standardized conditions with professional veterinary care and participate in a multidisciplinary program to assess the longitudinal dynamics of aging. The established Vaika infrastructure enables periodic gathering of quantitative data reflecting physical, physiological, immunological, neurological, and cognitive decline, as well as monitoring of aging-associated genetic and epigenetic alterations occurring in somatic cells. In addition, we assess the development of age-related diseases such as arthritis and cancer. In-depth data analysis, including artificial intelligence-based approaches, will build a comprehensive, integrated model of canine aging and potentially identify aging biomarkers that will allow use of this model for future testing of antiaging therapies.

Dog Ownership in Early Life Increased the Risk of Nonatopic Asthma in Children.

BACKGROUND: It is still debatable whether dog ownership during early childhood is a risk factor for the development of allergic diseases. OBJECTIVE: We investigated the association of dog ownership in early life with sensitization and asthma in childhood. METHODS: Data from the Cohort for Childhood Origin of Asthma and Allergic diseases were used to investigate the association between dog ownership at any time from pregnancy to 1 year of age and sensitization to aeroallergens at 3 and 7 years old, bronchial hyperresponsiveness (BHR), and asthma at 7 years old. We analyzed the cytokine levels in cord blood (CB) and indoor environmental measurement concentrations in the mother's residence obtained at 36 weeks of pregnancy. RESULTS: Sensitization to dogs at age 3 and 7 did not differ between dog ownership and nonownership, but dog ownership during early life decreased the risk of sensitization to aeroallergens at age 7 (aOR = 0.44, 95% CI 0.21-0.90). Dog ownership significantly increased the risk of nonatopic BHR (aOR = 2.86; 95% CI 1.32-6.21). In addition, dog ownership was associated with asthma, especially nonatopic asthma at 7 years old (aOR = 2.73, 95% CI 1.02-7.32; aOR = 7.05, 95% CI 1.85-26.90, respectively). There were no significant differences in the concentrations of IL-13 or interferon-γ in CB or indoor environmental measurements according to dog ownership during pregnancy. CONCLUSION: Early-life dog exposure in this birth cohort has been shown to reduce atopy but increase the risk of nonatopic BHR and nonatopic asthma at 7 years old.

Effect of Hydrolyzed Yeast Administration on Faecal Microbiota, Haematology, Serum Biochemistry and Cellular Immunity in Healthy Dogs.

Fungal microorganisms are regularly found in the gastrointestinal tract of healthy and diseased dogs especially from the phyla Ascomycota and Basidiomycota; however, it is necessary to better understand their role in host health. One of the most commonly studied yeast species in humans or animals is Saccharomyces cerevisiae especially in its live cell form. Scarce knowledge on its hydrolysate product effects in dogs forced us to test diet supplemented with hydrolyzed brewery S. cerevisiae (at a dose 0.3% of the diet) for 14 days to healthy adult dogs. Twenty German Shepherds were randomly divided into 2 groups: control and experimental, ten dogs in each. The experiment lasted 42 days (blood and faeces sample collection at days 0, 14, 28 and 42). The results of this straighforward experiment showed significant increase in the abundance of bifidobacteria (day 14), lactic acid bacteria (day 42) and clostridia (day 42). The faecal pH was significantly increased at day 28. In blood serum, the concentration of triglyceride and cholesterol decreased (day 42) while activities of alanine aminotransferase (at day 14) and aspartate aminotransferase significantly increased (at days 28 and 42). Activities of these enzymes were above reference range top in 7 dogs (ALT) and 4 dogs (AST). Haematological paramaters and activity of phagocytes as well as on percentage of lymphocyte subsets CD4+, CD8+, CD4+CD8+ and CD21+ were not changed during the experiment. The important point of these results is their onset mostly in the post-supplementation period. The observation of some unexpected effects emphasizes the need for reassessment to use yeasts products for dogs but further studies using different doses are necessary.

Array-based measurements of aero-allergen-specific IgE correlate with skin-prick test reactivity in asthma regardless of specific IgG4 or total IgE measurements.

Skin prick testing (SPT) and measurement of serum allergen-specific IgE (sIgE) are used to investigate asthma and other allergic conditions. Measurement of serum total IgE (tIgE) and allergen-specific IgG4 (sIgG4) may also be useful. The aim was to ascertain the correlation between these serological parameters and SPT. Sera from 60 suspected asthmatic patients and 18 healthy controls were assayed for sIgE and sIgG4 reactivity against a panel of 70 SPT allergen preparations, and for tIgE. The patients were also assessed by skin prick tests for reactivity to cat, dog, house dust mite and grass allergens. Over 50% of the patients had tIgE levels above the 75th percentile of the controls. 58% of patients and 39% of controls showed sIgE reactivity to ≥1 allergen. The mean number of allergens detected by sIgE was 3.1 in suspected asthma patients and 0.9 in controls. 58% of patients and 50% of controls showed sIgG4 reactivity to ≥1 allergen. The mean number of allergens detected by sIgG4 was 2.5 in patients and 1.7 in controls. For the patients, a strong correlation was observed between clinical SPT reactivity and serum sIgE levels to cat, dog, house dust mite (HDM) and grass allergens. SPT correlations using sIgE/sIgG4 or sIgE/tIgE ratios were not markedly higher. The measurement of serum sIgE by microarray using SPT allergen preparations showed good correlation with clinical SPT reactivity to cat, dog, HDM and grass allergens. This concordance was not improved by measuring tIgE or sIgG4.

Safety, biodistribution and viral shedding of oncolytic vaccinia virus TG6002 administered intravenously in healthy beagle dogs.

Oncolytic virotherapy is an emerging strategy that uses replication-competent viruses to kill tumor cells. We have reported the oncolytic effects of TG6002, a recombinant oncolytic vaccinia virus, in preclinical human xenograft models and canine tumor explants. To assess the safety, biodistribution and shedding of TG6002 administered by the intravenous route, we conducted a study in immune-competent healthy dogs. Three dogs each received a single intravenous injection of TG6002 at 105 PFU/kg, 106 PFU/kg or 107 PFU/kg, and one dog received three intravenous injections at 107 PFU/kg. The injections were well tolerated without any clinical, hematological or biochemical adverse events. Viral genomes were only detected in blood at the earliest sampling time point of one-hour post-injection at 107 PFU/kg. Post mortem analyses at day 35 allowed detection of viral DNA in the spleen of the dog which received three injections at 107 PFU/kg. Viral genomes were not detected in the urine, saliva or feces of any dogs. Seven days after the injections, a dose-dependent antibody mediated immune response was identified. In conclusion, intravenous administration of TG6002 shows a good safety profile, supporting the initiation of clinical trials in canine cancer patients as well as further development as a human cancer therapy.

Using data-driven approaches to improve delivery of animal health care interventions for public health.

Rabies kills ∼60,000 people per year. Annual vaccination of at least 70% of dogs has been shown to eliminate rabies in both human and canine populations. However, delivery of large-scale mass dog vaccination campaigns remains a challenge in many rabies-endemic countries. In sub-Saharan Africa, where the vast majority of dogs are owned, mass vaccination campaigns have typically depended on a combination of static point (SP) and door-to-door (D2D) approaches since SP-only campaigns often fail to achieve 70% vaccination coverage. However, D2D approaches are expensive, labor-intensive, and logistically challenging, raising the need to develop approaches that increase attendance at SPs. Here, we report a real-time, data-driven approach to improve efficiency of an urban dog vaccination campaign. Historically, we vaccinated ∼35,000 dogs in Blantyre city, Malawi, every year over a 20-d period each year using combined fixed SP (FSP) and D2D approaches. To enhance cost effectiveness, we used our historical vaccination dataset to define the barriers to FSP attendance. Guided by these insights, we redesigned our vaccination campaign by increasing the number of FSPs and eliminating the expensive and labor-intensive D2D component. Combined with roaming SPs, whose locations were defined through the real-time analysis of vaccination coverage data, this approach resulted in the vaccination of near-identical numbers of dogs in only 11 d. This approach has the potential to act as a template for successful and sustainable future urban SP-only dog vaccination campaigns.

Simultaneous allergic traits in dogs and their owners are associated with living environment, lifestyle and microbial exposures.

Both humans and pet dogs are more prone to develop allergies in urban than in rural environments, which has been associated with the differing microbial exposures between areas. However, potential similarities in the microbiota, that associate with environmental exposures, in allergic dogs and owners has not been investigated. We evaluated skin and gut microbiota, living environment, and lifestyle in 168 dog-owner pairs. Due to partly different manifestations of allergies between species, we focused on aeroallergen sensitized humans and dogs with owner-reported allergic symptoms. Our results agree with previous studies: dog-owner pairs suffered simultaneously from these allergic traits, higher risk associated with an urban environment, and the skin, but not gut, microbiota was partly shared by dog-owner pairs. We further discovered that urban environment homogenized both dog and human skin microbiota. Notably, certain bacterial taxa, which were associated with living environment and lifestyle, were also related with allergic traits, but these taxa differed between dogs and humans. Thus, we conclude that dogs and humans can be predisposed to allergy in response to same risk factors. However, as shared predisposing or protective bacterial taxa were not discovered, other factors than environmental microbial exposures can mediate the effect or furry dog and furless human skin select different taxa.

Gene expression of the immunoinflammatory and immunological status of obese dogs before and after weight loss.

Obesity is characterized by a low degree of chronic inflammation state that, along with metabolic modifications, promotes important changes in the animal's organism. Adipose tissue actively participates in inflammation and immunity, and several defense cells of the organism may, therefore, be involved in the diversity found between obese and ideal weight individuals. Studies regarding this subject have shown immune cell changes in humans and rats, however, the literature is scarce in relation to dogs. Thus, the present study aimed to evaluate the gene expression profile of immunoinflammatory response and the lymphoproliferation of obese dogs before and after weight loss. Eight female dogs, neutered, of different breeds, aged between 1 and 8 years (4.74±3.19), obese, with body condition score (BCS) of 9 out of a 9-point scale and body composition determined by the deuterium isotope dilution method were included. The obese dogs were enrolled in a weight loss program and after losing 20% of their initial weight became a second experimental group. A third experimental group consisted of eight female dogs, neutered, aged between 1 and 8 years (3.11±0.78) and with ideal BCS (5 out of a 9-point scale). Gene expression of immunoinflammatory cytokines (resistin, leptin, adiponectin, TNF-α, IL-6, IL-8, and IL-10) was assessed by qRT-PCR and immunity was assessed by lymphoproliferative response using the flow cytometry technique. The data that presented normal distribution was evaluated by analysis of variance by the PROC MIXED of the SAS and when differences were detected, these were compared by the Tukey test. Regarding the gene expression data, the procedure PROC GLIMMIX was adopted and the methodology of generalized linear model was used, in which the Gama distribution proved to be adequate. Values of p<0.05 were considered significant. The mean weight loss period of the animals included in the study was 194.25 ± 28.31 days and the mean weekly weight loss rate was 1.02 ± 0.82%. The average fat mass, both in percentage (P<0.001) and in kilograms (P = 0.012), was higher in the obese group (40.88%; 8.91kg), returning to normal and without difference between the control group (19.16%; 3.01kg) and after weight loss (22.10%; 4.11kg). The weight loss program resulted in an increase in percentage of lean body mass (P = 0.001), 55.50% in obese animals vs 77.90% in obese dogs after weight loss, the latter with no difference when compared to the control group (80.84%). The obese group presented increased gene expression of resistin and IL-8 in relation to the weight loss group (P = 0.002). In adiponectin, the obese group presented increased mRNA gene expression when compared to the weight loss group (P = 0.003). The evaluation of lymphocyte proliferation showed differences between the group of obese animals before and after weight loss (P = 0.004). Weight loss resulted in an increase in the lymphoproliferation rate (18.48%) compared to obese dogs at the beginning of the study (10.71%). These results indicate that weight loss modulates the immunoinflammatory response of obese dogs and may present important benefits to health and longevity of dogs.

Profile of gamma-delta (γδ) T lymphocytes in the peripheral blood of crossbreed dogs during stages of life and implication in aging.

BACKGROUND: Data on gamma-delta (γδ) T lymphocytes in the peripheral blood of dogs are scant, related only to healthy pure breed dogs and limited to a restricted age range. The aim of the study was to investigate the modulation of the γδ T lymphocyte (TCRγδ+) subpopulation in peripheral blood of crossbreed healthy dogs according to five identified stages of life: Puppy, Junior, Adult, Mature, Senior and to determine its implication in aging. A rigorous method of recruitment was used to minimize the influence of internal or external pressure on the immune response. Twenty-three intact female and twenty-four intact male dogs were enrolled. Blood samples were collected and immunophenotyping of peripheral blood T lymphocytes and γδ T cell subpopulations was performed. RESULTS: The percentage of γδ T cells in peripheral blood lymphocytes was comparable with the value of 2.5% published by Faldyna and co-workers (2001), despite the percentage reported was investigated in less arranged age range groups and coming from four different dog pure breeds, whereas our data were recorded on wider age range groups and coming from crossbreed dogs. Therefore, the γδ T cell percentage (2.5%) is consistent and points out that such value is breed-independent. Statistical analysis highlighted differences in both percentage and absolute γδ T cells according to the stage of life. γδ T cells decreased significantly in the peripheral blood of elder dogs (Senior group) in comparison with previous stages of life (Puppy, Junior, and Adult groups). Differences in γδ T cells are significant and they are reported, for the first time, related to dog aging. CONCLUSIONS: The study confirms dogs to be among the animals with a low TCRγδ+ cell profile. A decrease of the TCRγδ+ subpopulation percentage was observed in elder dogs. TCRγδ+ cells of group S were different from those of groups P, J, and A. The differences are reported for the first time in dog aging. Identifying the stage of life when the decrease of γδ T lymphocytes starts can be useful for providing a rationale for drafting a wellness plan trial to support thymus immune functions and mitigate its functional exhaustion.

The utility of DLA typing for transplantation medicine in canine models.

Transplantation medicine is used for the treatment of severe canine diseases, and the dog leukocyte antigen (DLA) is considered to be important in graft rejection. However, the utility of direct sequencing of both DLA classes I and II has not been assessed thoroughly. Eight healthy beagles with identified DLA genes were divided into two sets of four dogs, each including one donor and three recipients for skin transplantation. The following recipients were selected: one dog with a complete match, one with a haploidentical match, and one with a complete mismatch of the DLA gene with the donor. Full-thickness skin segments were obtained from each donor and transplanted to the recipients. A mixed lymphocyte reaction (MLR) assay was performed and analyzed by flow cytometry. Skin grafts of DLA haploidentical and mismatched pairs were grossly rejected within 14 days, whereas in fully matched DLA pairs, survival was as long as 21 days. Histopathological evaluation also showed moderate to severe lymphocytic infiltration and necrosis in DLA mismatched pairs. As seen in the MLR assay, the stimulation index of DLA mismatched pairs was significantly higher than that of fully matched DLA pairs in both sets (P<0.001). The allogeneic transplantation results suggested that it is possible to prolong transplant engraftment by completely matching the DLA genotype between the donor and recipient. Additionally, the MLR assay may be used as a simplified in vitro method to select donors.

Aspectos clínicos, laboratoriais e tratamento da leucemia mieloide crônica em cão: relato de caso / Clinical aspects, hematological changes and treatments of chronic myeloid leukemia in dog: case report

O objetivo deste relato é apresentar o caso clínico de uma cadela, sem raça definida, com cinco anos de idade, diagnosticada com leucemia mieloide crônica (LMC). As leucemias são neoplasias malignas que se originam de células precursoras da medula óssea e as consequências podem ser trombocitopenia, anemia, leucocitose persistente e presença de células neoplásicas no sangue. O tratamento de escolha envolve o uso de inibidores de tirosina quinase, porém este não pode ser usado neste caso. Dessa forma a cadela recebeu diferentes protocolos quimioterápicos que incluíram inicialmente hidroxiureia, citarabina, doxorrubicina e prednisona. Devido a remissão parcial dos sinais clínicos e a resposta terapêutica pouco duradoura a essas medicações o protocolo foi alterado para quimioterapia metronômica com clorambucil. O uso desses quimioterápicos não foram eficazes em reduzir a leucocitose e controlar a anemia e trombocitopenia da paciente, devido a ocorrência do surgimento de células imaturas no sangue e resistência aos quimioterápicos. Na ausência da crise e da possibilidade do uso dos inibidores de tirosina quinase, a hidroxiureia permanece sendo o quimioterápico de eleição. O animal apresentou sobrevida de 210 dias, devido a leucocitose e anemia severas pouco responsivas ao protocolo terapêutico utilizado e o surgimento no hemograma de precursores neutrofilicos que ocorreu 46 dias após ao início do tratamento com hidroxiureia.

The aim of this report is to present the clinical case of a five-year-old mixed breed female dog diagnosed with chronic myeloid leukemia (CML). Leukemias are malignant neoplasms that originate from bone marrow precursor cells and the consequences can be thrombocytopenia, anemia, persistent leukocytosis and the presence of neoplastic cells in the blood. The treatment of choice involves the use of tyrosine kinase inhibitors, but it cannot be used in this case. Thus, the dog received different chemotherapy protocols that initially included hydroxyurea, cytarabine, doxorubicin and prednisone. Due to the partial remission of clinical signs and the short-term therapeutic response to these medications, the protocol was changed to metronomic chemotherapy with chlorambucil. The use of these chemotherapeutic agents was not effective in reducing leukocytosis and controlling the patient's anemia and thrombocytopenia, due to the occurrence of immature cells in the blood and resistance to chemotherapeutic agents. In the absence of the crisis and the possibility of using tyrosine kinase inhibitors, hydroxyurea remains the chemotherapy of choice. The animal had a 210-day survival, due to severe leukocytosis and anemia, which were not responsive to the therapeutic protocol used and the appearance in the blood count of neutrophilic precursors that occurred 46 days after the beginning of hydroxyurea treatment.

Transportation and Routine Veterinary Interventions Alter Immune Function in the Dog.

Rapid activation of the hypothalamic pituitary adrenal axis and the sympathetic nervous system are hallmarks of the acute stress response and these systems interact with the immune system by signaling though glucocorticoid and adrenergic receptors on immune cells. There is limited information about the effect of these physiologic responses on immunologic parameters of pet dogs enrolled in clinical studies. The objective of this study was to evaluate how travel, instrumentation, and hospitalization alter immunologic parameters in pet dogs. Blood was collected from healthy dogs in a home environment and from healthy dogs at the time of presentation to the hospital and after instrumentation and 24 hours of hospitalization. We found that lipopolysaccharide (LPS)-induced downregulation of toll like receptor 4 (TLR4) was blunted in dogs exposed to stress. Neutrophil and monocyte major histocompatibility complex class II (MHCII) expression increased after transportation to the veterinary hospital but then became similar to that of the control dogs at the end of hospitalization. Peripheral blood mononuclear cell cytotoxicity function was blunted in dogs exposed to the stress of transportation as well as hospitalization. Neutrophil apoptosis was greater in dogs exposed to stress compared to controls although this effect significantly decreased after hospitalization stress. Conversely, stress did not alter induced or spontaneous cytokine production from leukocytes, neutrophil or monocyte expression of TLR4, LPS-induced downregulation of monocyte TLR4, LPS-induced neutrophil and monocyte expression of MHCII or peripheral blood lymphocyte phenotype. Transportation and instrumentation/hospitalization stress should be considered when interpreting immunologic studies in pet dogs.

Teriflunomide inhibits activation-induced CD25 expression on T cells and may affect Foxp3-expressing regulatory T cells.

Teriflunomide (TER) is an immunomodulatory agent. Although the first reports on the use of TER in dogs have already appeared, immune mechanisms underlying the immunomodulatory effect of TER do not seem to have been fully elucidated yet. There were two aspects of this study. First, further insight into the mode of action of TER was gained by investigating its effect on the expression of IL-2 receptor α-chain (CD25) and Forkhead box P3 (Foxp3) by CD4+ and CD8+ T cells and apoptosis of these cells. Second, in view in the earlier lack of data on the effect of TER on T cells in dogs, the results of this study filled in this gap. TER at a concentration which can be achieved in vivo prevented or reduced the activation-induced CD25 expression on CD4+ and CD8+ T cells, respectively. Taking into consideration the role of CD25 in T cell proliferation, this effect may constitute an additional mechanism responsible for the antiproliferative effect of the drug. Under stimulation conditions, TER induced Foxp3 expression in Foxp3-negative CD4+ and CD8+ T cells, while down-regulating it under unstimulated conditions. These results suggest that TER may generate iTreg cells, but this process requires cell activation. TER was not found to affect on the absolute count and apoptosis of CD4+ and CD8+ T cells. The results suggest that the impairment of CD25 expression during T cell activation and generation of iTreg cells may constitute additional mechanisms, besides the principal one, underlying the immunomodulatory effect of TER.

Prevalence of Dal blood type and dog erythrocyte antigens (DEA) 1, 4, and 7 in canine blood donors in Italy and Spain.

BACKGROUND: The aim of this study was to determine the prevalence of Dal, and DEA 1, 4, 7 blood types, in a population of canine blood donors from Italy and Spain. Three hundred and twenty blood donor dogs receiving an annual health evaluation were included in the study. DEA 1 blood type was determined using an immunochromatographic strip technique while Dal, DEA 4 and 7 blood types were determined with polyclonal antisera using agglutination on gel columns. RESULTS: Out of 320 dogs blood typed 7 (2 Cane Corso and 5 Doberman Pinschers) (2.2%) were Dal negative; 137 (42.8%) were positive for DEA 1; 320 (100%) were positive for DEA 4 and 43 (13.4%) were positive for DEA 7. CONCLUSION: This study showed a similar prevalence of DEA 1, 7 and 4 to that reported in previous studies in the same, and in different, geographic areas, and provides new data on the prevalence of the Dal blood group in Italy and Spain. There was no significant difference (P = 0.8409) between prevalence of Dal negative blood types found in our population (2.2%) and the prevalence reported in a canine blood donor population from the USA (2.5%). Our study identified Dal negative dogs in a previously tested breed i.e. Doberman Pinschers, but also the Cane Corso breed was found to have Dal negative dogs.

Effects of educational efforts in tribal homes and schools to reduce asthma triggers, symptoms and missed school days.

Native American asthma prevalence has been estimated higher than for the U.S. population average, and uncontrolled asthma results in absence from schools. This study analyzed effects of targeted education campaigns in both homes and schools on reducing asthma triggers and symptoms among 119 children with asthma, who were recruited from Cherokee and Nez Perce communities. The education campaigns were developed in collaboration with the researchers and the tribes, tailoring strategies to reduce asthma triggers adapted to tribal lifestyles. There was a special emphasis in identification of Traditional Ecological Knowledge (TEK) aspects, which were woven into the study plan and implementation. Some 62 study homes were investigated and the parents received targeted education at the beginning of the study, whereas the remaining control homes received the same education at the end of the study. In Cherokee homes, allergen levels were significantly increased in both control and study groups throughout the study. In Nez Perce homes, dog and cockroach allergen levels decreased significantly in the study homes. The parents reported asthma control test (ACT) scores of the children were significantly improved in both study and control groups, whereas respiratory illness days were reduced in the study group. In schools, allergen levels, particularly cat, dog and cockroach allergens, increased throughout the year in both study and control schools. However, high contact surface cleaning effectiveness based on adenosine tri phosphate (ATP) readings was improved in the study schools. It was also found that high contact surface cleaning effectiveness may have effects on students' absence rates tracked by schools. In conclusion, the results indicate partial improvements in parent reported health symptoms, although the improvements could not be definitively attributed to reduction of any specific exposure in the home environment. In the school environment, effective cleaning throughout the flu season could result in decreased absence rates.

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.

Establishment of rat anti-canine DEP domain containing 1B (DEPDC1B) monoclonal antibodies.

DEP domain-containing 1B (DEPDC1B) is involved in the regulation of cell de-adhesion and actin cytoskeleton activity during the G2/M transition of the cell cycle, and its overexpression has been proven to be associated with cancer progression in several human cancers. Canine DEPDC1B was identified as a gene that was overexpressed in canine lymphoma tissues in our previous study. However, in dogs, the protein expression of DEPDC1B remains to be determined due to the lack of a specific monoclonal antibody. Here, we developed rat monoclonal antibodies against canine DEPDC1B and characterized their applicability for immunodetection assays. Our findings demonstrated that these antibodies are functional and can be important tools to investigate the precise role of DEPDC1B in canine tumors.