Laboratory indicators of hypothyroidism and TgAA-positivity in the Eurasian dog breed.

BACKGROUND: Hereditary hypothyroidism represents a concern for dog breeders; thus, surveillance programs have been established for several dog breeds. METHODS: Thyroid profiles (total thyroxine (TT4), thyrotropin (thyroid stimulating hormone (TSH)), and thyroglobulin autoantibodies (TgAA)) collected as part of a breed surveillance program in Eurasians (2009-2017) were retrospectively analyzed. The study included data from 1,501 Eurasians from a German breeding club. Classification was exclusively based on laboratory data. Hypothyroidism was defined as a combined decrease in TT4 and increase in TSH in serum and was classified as TgAA-positive and TgAA-negative hypothyroidism. Thyroglobulin autoantibodies (TgAA) independent of the concentrations of TT4 and TSH were determined. The overall prevalence of hypothyroidism, TgAA-positive hypothyroidism, TgAA-negative hypothyroidism and TgAA-positivity was assessed when the dogs entered the program. Follow-up laboratory data was available for 324 dogs without hypothyroidism on initial examination. RESULTS: The initial screening was performed at a median age of 18 months (interquartile range (IQR): 15-29). The overall prevalence of hypothyroidism was 3.9% (n = 58; 95% CI: 2.9-4.8%) and the prevalence of a positive TgAA status was 7.9% (n = 118; 95% CI: 6.6-9.3%). The prevalence of TgAA-positive and TgAA-negative hypothyroidism was 1.7% (n = 26; 95% CI: 1.1-2.4%) and 2.1% (n = 32; 95% CI: 1.4-2.9%), respectively. 22.0% of dogs with positive TgAA status (26/118) were already hypothyroid on initial examination. Overall, 42.5% (17/40) of TgAA-positive dogs on initial examination developed hypothyroidism on follow-up. CONCLUSION: The results of this study demonstrate that the Eurasian dog breed exhibits a relevant risk for hypothyroidism and presence of TgAA. The predictive value of TgAA for hypothyroidism or developing hypothyroidism was high in this breed. Further investigations with longitudinal studies in individual dogs are warranted.

Expression of CD9 on porcine lymphocytes and its relation to T cell differentiation and cytokine production.

In this work, we report on two novel monoclonal antibodies, specific for porcine CD9. CD9 is a tetraspanin that is expressed on a wide variety of cells. We phenotyped porcine immune cell subsets and found that CD9 was expressed on all monocytes as well as a subset of B cells. CD9 was variably expressed on T cells, with CD4 T cells containing the highest frequency of CD9+ cells. CD9 expression positively correlated with the frequency of central memory CD4 T cells in ex vivo PBMC. Therefore, we proceeded to explore CD9 as a marker of T cell function. Here we observed that CD9 was expressed on the vast majority of long-lived influenza A virus-specific effector cells that retained the capacity for cytokine production in response to in vitro recall antigen. Therefore, the new antibodies enable the detection of a cell surface molecule with functional relevance to T cells. Considering the importance of CD9 in membrane remodelling across many cell types, they will also benefit the wider field of swine biomedical research.

The Effects of Play Behavior, Feeding, and Time of Day on Salivary Concentrations of sIgA in Calves.

The focus of animal welfare science has shifted over the last decades from efforts to avoid negative states to ways of allowing animals the experience of positive emotions. They may influence physiological processes in farmed animals, potentially providing health benefits; in addition, the physiological changes might be used as indicators of emotional states. We investigated calves' salivary secretory immunoglobulin A (sIgA) concentrations with regard to a possible circadian rhythm and two situations that elicit positive emotions. Ten saliva samples of 14 calves were taken on two consecutive days; within the course of a day we observed a significant decline in salivary sIgA concentrations at 14:00 h. Further, we probed the animals before and after milk feeding and, contrarily to our prediction, detected lower sIgA concentrations 5 min after feeding than 15 min before. A probable explanation might be an increase in salivary flow rate caused by milk ingestion. We also took samples before and after we stimulated play behavior in calves. There was no significant difference in sIgA concentrations between samples taken before and after play. Although there was a significant correlation between the change in sIgA concentrations and the amount of play behavior shown, the correlation depended on an unexpected decrease of sIgA in animals that played little, and thus, does not support our hypothesis. In general, the data showed a large variability that might arise from different factors that are difficult to standardize in animals. Thus, the use of salivary sIgA concentrations as a marker of positive emotions in calves is not supported conclusively by the present data.

An Alliance of Gel-Based and Gel-Free Proteomic Techniques Displays Substantial Insight Into the Proteome of a Virulent and an Attenuated Histomonas meleagridis Strain.

The unicellular protozoan Histomonas meleagridis is notorious for being the causative agent of histomonosis, which can cause high mortality in turkeys and substantial production losses in chickens. The complete absence of commercially available curative strategies against the disease renders the devising of novel approaches a necessity. A fundamental step toward this objective is to understand the flagellate's virulence and attenuation mechanisms. For this purpose we have previously conducted a comparative proteomic analysis of an in vitro cultivated virulent and attenuated histomonad parasite using two-dimensional electrophoresis and MALDI-TOF/TOF. The current work aimed to substantially extend the knowledge of the flagellate's proteome by applying 2D-DIGE and sequential window acquisition of all theoretical mass spectra (SWATH) MS as tools on the two well-defined strains. In the gel-based experiments, 49 identified protein spots were found to be differentially expressed, of which 37 belonged to the in vitro cultivated virulent strain and 12 to the attenuated one. The most frequently identified proteins in the virulent strain take part in cytoskeleton formation, carbohydrate metabolism and adaptation to stress. However, post-translationally modified or truncated ubiquitous cellular proteins such as actin and GAPDH were identified as upregulated in multiple gel positions. This indicated their contribution to processes not related to cytoskeleton and carbohydrate metabolism, such as fibronectin or plasminogen binding. Proteins involved in cell division and cytoskeleton organization were frequently observed in the attenuated strain. The findings of the gel-based studies were supplemented by the gel-free SWATH MS analysis, which identified and quantified 42 significantly differentially regulated proteins. In this case proteins with peptidase activity, metabolic proteins and actin-regulating proteins were the most frequent findings in the virulent strain, while proteins involved in hydrogenosomal carbohydrate metabolism dominated the results in the attenuated one.

Unravelling the differences: comparative proteomic analysis of a clonal virulent and an attenuated Histomonas meleagridis strain.

The current study focused on Histomonas meleagridis, a unicellular protozoan, responsible for histomonosis in poultry. Recently, the occurrence of the disease increased due to the ban of effective chemotherapeutic drugs. Basic questions regarding the molecular biology, virulence mechanisms or even life cycle of the flagellate are still puzzling. In order to address some of these issues, we conducted a comparative proteomic analysis of a virulent and an attenuated H. meleagridis strain traced back to a single cell and propagated in vitro as monoxenic mono-eukaryotic cultures. Using two-dimensional electrophoresis (2-DE) for proteome visualization with computational 2-DE gel image and statistical analysis, upregulated proteins in either of the two H. meleagridis strains were detected. Statistical analysis fulfilling two criteria (≥threefold upregulation and P < 0.05) revealed 119 differentially expressed protein spots out of which 62 spots were noticed in gels with proteins from the virulent and 57 spots in gels with proteins from the attenuated culture. Mass spectrometric analysis of 32 protein spots upregulated in gels of the virulent strain identified 17 as H. meleagridis-specific. The identification revealed that these spots belonged to eight different proteins, with the majority related to cellular stress management. Two ubiquitous cellular proteins, actin and enolase, were upregulated in multiple gel positions in this strain, indicating either post-translational modification or truncation, or even both. Additionally, a known virulence factor named legumain cysteine peptidase was also detected. In contrast to this, mass spectrometric analysis of 49 protein spots, upregulated in gels of the attenuated strain, singled out 32 spots as specific for the flagellate. These spots were shown to correspond to 24 different proteins that reflect the increased metabolism, in vitro adaptation of the parasite, and amoeboid morphology. In addition to H. meleagridis proteins, the analysis identified differential expression of Escherichia coli DH5α proteins that could have been influenced by the co-cultivated H. meleagridis strain, indicating a reciprocal interaction of these two organisms during monoxenic cultivation.

Host-pathogen interplay at primary infection sites in pigs challenged with Actinobacillus pleuropneumoniae.

BACKGROUND: Actinobacillus (A.) pleuropneumoniae is the causative agent of porcine pleuropneumonia and causes significant losses in the pig industry worldwide. Early host immune response is crucial for further progression of the disease. A. pleuropneumoniae is either rapidly eliminated by the immune system or switches to a long-term persistent form. To gain insight into the host-pathogen interaction during the early stages of infection, pigs were inoculated intratracheally with A. pleuropneumoniae serotype 2 and humanely euthanized eight hours after infection. Gene expression studies of inflammatory cytokines and the acute phase proteins haptoglobin, serum amyloid A and C-reactive protein were carried out by RT-qPCR from the lung, liver, tonsils and salivary gland. In addition, the concentration of cytokines and acute phase proteins were measured by quantitative immunoassays in bronchoalveolar lavage fluid, serum and saliva. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. RESULTS: Significant cytokine and acute phase protein gene expression was detected in the lung and the salivary gland however this was not observed in the tonsils. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter investigations, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. The bacteria isolated from the upper and lower respiratory tract showed distinct IR spectral patterns reflecting the organ-specific acute phase response of the host. CONCLUSIONS: In summary, this study implies a metabolic adaptation of A. pleuropneumoniae to the porcine upper respiratory tract already during early infection, which might indicate a first step towards the persistence of A. pleuropneumoniae. Not only in lung, but also in the salivary gland an increased inflammatory gene expression was detectable during the acute stage of infection.

IL-12 and IL-18 induce interferon-γ production and de novo CD2 expression in porcine γδ T cells.

γδ T cells are highly abundant in the blood and spleen of pigs but little is known about their functional differentiation. In this study the potential of the type-1 polarizing cytokines IL-12 and IL-18 in combination with IL-2 and Concanavalin A (ConA) to stimulate porcine γδ T cells was investigated. Stimulation of purified γδ T cells with ConA and IL-2 induced a strong proliferation of CD2(-) γδ T cells, whereas additional stimulation with IL-12 and IL-18 caused a stronger proliferation of CD2(+) γδ T cells. IFN-γ could only be detected in supernatants of γδ T-cell cultures supplemented with IL-12 and IL-18. Experiments with sorted CD2/SWC5-defined γδ T-cell subsets revealed that CD2(+)SWC5(-) γδ T cells are the main producers of IFN-γ following stimulation with IL-2/IL-12/IL-18. Additional stimulation with ConA led to an upregulation of CD2 within the CD2(-) γδ T cell subsets, indicating a previously unnoticed plasticity of CD2-defined γδ T cell subsets.

CD2 and CD8α define porcine γδ T cells with distinct cytokine production profiles.

γδ T cells are a remarkably prominent T-cell subset in swine with a high prevalence in blood. Phenotypic analyses in this study showed that CD2(-) γδ T cells in their vast majority had a CD8α(-)SLA-DR(-)CD27(+) phenotype. CD2(+) γδ T cells dominated in spleen and lymph nodes and had a more heterogeneous phenotype. CD8α(+)SLA-DR(-)CD27(+) γδ T cells prevailed in blood, spleen and lymph nodes whereas in liver a CD8α(+)SLA-DR(+)CD27(-) phenotype dominated, indicating an enrichment of terminally differentiated γδ T cells. γδ T cells were also investigated for their potential to produce IFN-γ, TNF-α and IL-17A. Within CD2(+) γδ T cells, IFN-γ and TNF-α single-producers as well as IFN-γ/TNF-α double-producers dominated, which had a CD8α(+)CD27(+/-) phenotype. IL-17A-producing γδ T cells were only found within CD2(-) γδ T cells, mostly co-produced TNF-α and had a rare CD8α(+)CD27(-) phenotype. However, quantitatively TNF-α single-producers strongly dominated within CD2(-) γδ T cells. In summary, our data identify CD2 and CD8α as important molecules correlating with functional differentiation.

Porcine CD27: identification, expression and functional aspects in lymphocyte subsets in swine.

Up to now for Swine Workshop Cluster 2 (SWC2) the orthologous human CD molecule was unknown. By use of the SWC2-specific mAb b30c7 and a retroviral cDNA expression library derived from stimulated porcine peripheral blood mononuclear cells we could identify SWC2 as porcine CD27. Phenotypic analyses of lymphocytes isolated from blood and lymphatic organs revealed that mature T cells in thymus and T cells in the periphery with a naïve phenotype were CD27(+). However, within CD8α(+) T helper and CD8α(+) γδ T cells also CD27(-) cells were present, indicating a down-regulation after antigen contact in vivo. B cells lacked CD27 expression, whereas NK cells expressed intermediate levels. Furthermore, plate-bound mAb b30c7 showed a costimulatory capacity on CD3-activated T cells for proliferation, IFN-γ and TNF-α production. Hence, our data indicate an important role of porcine CD27 for T-cell differentiation and activation as described for humans and mice.

Cysteine peptidases, secreted by Trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture.

Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.

NKp46 expression discriminates porcine NK cells with different functional properties.

So far little is known about natural killer (NK) cells in the pig due to the lack of NK cell-specific markers. In this study, we identified the activating receptor NKp46 (CD335) in swine with newly developed monoclonal antibodies (mAbs) for more detailed studies on NK cells in this species. The NKp46 mAbs showed a specific reactivity with a distinct population of perforin(+) CD2(+) CD3(-) CD8α(+) CD16(+) lymphocytes. In spleen and liver, an additional subset of CD8α(dim/-) lymphocytes with increased NKp46 expression was observed. Surprisingly, we could identify NKp46(-) cells with an NK cell phenotype in all animals analyzed. These lymphocytes showed comparable cytolytic activity against xenogeneic and allogeneic target cells as NKp46(+) NK cells. In contrast, NKp46(+) NK cells produced several fold higher levels of interferon-γ (IFN-γ) than the NKp46(-) cells after cytokine stimulation. Furthermore, an activation-dependent induction of NKp46 expression in formerly NKp46(-) cells after stimulation with interleukin-2 (IL-2), IL-12, and IL-18 could be shown. In summary, our data indicate that NKp46 is not expressed by all porcine NK cells and that NKp46 discriminates porcine NK cells differing in regard to cytokine production, which challenges the paradigm of NKp46 as a comprehensive marker for NK cells across different mammalian species.

Current knowledge on porcine regulatory T cells.

Regulatory T cells (Tregs) are known in humans and mice from last 15 years, and several studies led to a detailed knowledge on their phenotype, functions, and role in various immune reactions. In swine, the existence of Tregs was first demonstrated in 2008 and research is still at the beginning. Nevertheless, basic information regarding phenotype, mechanisms and targets of suppression, as well as implications in transplantation and some diseases are available. Purpose of this review is to give a brief summary of the current knowledge about porcine Tregs.

Effect of radiation on vascular endothelial growth factor expression in the C2 canine mastocytoma cell line.

OBJECTIVE: To establish the radiosensitivity and effect of irradiation on vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) expression in the canine mastocytoma cell line C2. SAMPLE POPULATION: Canine mastocytoma cell line C2. PROCEDURES: C2 cells were irradiated with single doses of 2, 4, 6, and 8 Gy. The 3-(4, 5-di-methyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and proliferation assays with (methyl-hydrogen 3) thymidine were used for radiosensitivity experiments. Expression of VEGFR was determined via flow cytometry and apoptotic rate via annexin assay. Human and canine VEGF ELISA kits were evaluated in crossover assay experiments, and the canine kit was used thereafter. RESULTS: C2 cells secreted VEGF constitutively. Radiation did not induce a significant increase in VEGF secretion, regardless of radiation dose. Consistently, radiation did not up-regulate VEGFR. Cell survival rates decreased in a dose-dependent manner. The apoptotic cell fraction had a dose-dependent increase that reached its maximum 24 to 48 hours after radiation. CONCLUSIONS AND CLINICAL RELEVANCE: The C2 cell line was radiosensitive, and a fraction (up to 40%) of cells died via apoptosis in a dose-dependent manner. In response to radiation, C2 cells did not upregulate VEGF production or VEGFR. Further studies are needed to determine whether tumor control could be improved by combining radiotherapy with VEGFR inhibitors or apoptosis-modulating agents.

Detection of Foxp3 protein expression in porcine T lymphocytes.

Regulatory T cells (Tregs) are potent regulators of various immune reactions. Due to the lack of Treg-specific markers their analysis had often been challenging until the discovery of the transcription factor Forkhead-box p3 (Foxp3) which serves as this highly demanded marker. So far, antibodies staining human and murine Foxp3 have been developed. This study describes the analysis of four commercially available anti-Foxp3 antibodies for reactivity with their specific antigen in cells derived from porcine lymphoid tissues. One out of the four antibodies showed selective reactivity with porcine CD25(+) T lymphocytes. The intracellular antigen was expressed on a small subset of CD25(dim) cells and the majority of the CD25(high) positive T-cell subpopulation. Intracellular antigen positive cells showed a heterogeneous expression of other leukocyte differentiation antigens. The majority belonged to the CD4(+)CD8(+) T-lymphocyte subpopulation, but were also found in the CD4(+)CD8(-) subpopulation. Another small minority was included in the CD4(-)CD8(+) T-lymphocyte subpopulation. Additionally, a small fraction of the putative Foxp3(+) cells showed an expression of MHC-II molecules. These staining patterns in three and four colour flow cytometry analyses indicated that the cells detected by a rat anti-mouse/rat-Foxp3 antibody expressed the porcine Foxp3. The expression of the putative Foxp3 protein in distinct leukocyte subsets was confirmed by molecular analysis of Foxp3 mRNA expression. Using Western blot analysis specific protein bands could only be detected in fractions that also exhibited the corresponding Foxp3 mRNA expression. These experiments also revealed that the antibody recognized a single chain protein with a molecular mass of about 45kDA similar to Foxp3 described for other species. In summary, these data strongly indicate the reactivity of this antibody with porcine Foxp3. Thereby, this rat anti-mouse/rat Foxp3 antibody presents a powerful tool for the identification of porcine regulatory T cells.

Phenotypic and functional characterisation of porcine CD4(+)CD25(high) regulatory T cells.

Over the last years regulatory T cells (Tregs) were defined as CD4(+)CD25(+) T lymphocytes expressing the transcription factor Foxp3 (Forkhead Box P3) with the ability to downregulate various immune responses. In swine the existence of CD4(+)CD25(+) T lymphocytes was described before but nothing is known about the function of this minor cell population to date. Therefore, we studied porcine CD4(+)CD25(+) T cells with regard to major attributes of murine and human Tregs: their phenotype concerning the expression of several T-cell specific antigens, including Foxp3, their IL-10 production and their suppressive capacity. Our results revealed that porcine CD4(+)CD25(+) T cells with high CD25 expression count for about 2-9% of the CD4(+) T-cell subset. They demonstrate a strong Foxp3 expression, a heterogeneous CD45RC-, CD8alpha- and MHC-II-defined phenotype and a moderate IL-10 production. Co-cultivation of increasing numbers of CD4(+)CD25(high) T cells with a constant number of CD4(+)CD25(-) responder-T cells caused a decrease in proliferation of the entire culture. This demonstrates the suppressive capacity of the CD4(+)CD25(high) T-cell subset and - together with their Foxp3 expression - the existence of porcine Tregs.

Detection of autoantibodies against thyroid peroxidase in serum samples of hypothyroid dogs.

OBJECTIVE: To establish a sensitive test for the detection of autoantibodies against thyroid peroxidase (TPO) in canine serum samples. SAMPLE POPULATION: 365 serum samples from dogs with hypothyroidism as determined on the basis of serum concentrations of total and free triiodothyronine (T3), total and free thyroxine (T4), and thyroid-stimulating hormone, of which 195 (53%) had positive results for at least 1 of 3 thyroid autoantibodies (against thyroglobulin [Tg], T4, or T3) and serum samples from 28 healthy dogs (control samples). PROCEDURE: TPO was purified from canine thyroid glands by extraction with detergents, ultracentrifugation, and precipitation with ammonium sulfate. Screening for anti-TPO autoantibodies in canine sera was performed by use of an immunoblot assay. Thyroid extract containing TPO was separated electrophoretically, blotted, and probed with canine sera. Alkaline phosphatase-conjugated rabbit anti-dog IgG was used for detection of bound antibodies. RESULTS: TPO bands were observed at 110, 100, and 40 kd. Anti-TPO autoantibodies against the 40-kd fragment were detected in 33 (17%) sera of dogs with positive results for anti-Tg, anti-T4, or anti-T3 autoantibodies but not in sera of hypothyroid dogs without these autoantibodies or in sera of healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The immunoblot assay was a sensitive and specific method for the detection of autoantibodies because it also provided information about the antigen. Anti-TPO autoantibodies were clearly detected in a fraction of hypothyroid dogs. The value of anti-TPO autoantibodies for use in early diagnosis of animals with thyroid gland diseases should be evaluated in additional studies.