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

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

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

BackgroundHereditary hypothyroidism represents a concern for dog breeders; thus, surveillance programs have been established for several dog breeds.MethodsThyroid 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.ResultsThe 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.ConclusionThe 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

Results of the MS homology search for the protein Spot 8.

<p>The top 10 matches from the search are displayed. Following parameters were used for search: taxonomy search in <i>Trichomonas vaginalis</i>; Database searched: UniProtKB.2011.01.11; Mass Tolerance: 0.5 Da; Digest Used: No enzyme; Max. # Missed Cleavages: 1; Min matches: 1; Score matrix: BLOSUM62; List of Peptide Sequences: 1 FSY [I|L]ADYPYTAR 3, 2 NYW [I|L]VR 1, 3 NSWGASWGEK 2 (number left to the peptide sequence is used as the name of the peptide, whereas the number right to the peptide sequence refers to the maximal number of allowed mismatches). Mismatched amino acids are shown in bold.</p

Results of the MS homology search for the peptide m/z 1466.

<p>All matches selected by MS homology search are displayed. Following parameters were used for search: Taxonomy search in <i>Trichomonas vaginalis</i>; Database searched: UniProtKB.2011.01.11; Mass Tolerance: 0.5 Da; Digest Used: No enzyme; Max. # Missed Cleavages: 1; Min matches: 1; Score matrix: BLOSUM62; Peptide Sequence: 1 FSY [I|L]ADYPYTAR 3 (number left to the peptide sequence is used as the name of the peptide, whereas the number right to the peptide sequence refers to the maximal number of allowed mismatches). Mismatched amino acids are shown in bold.</p

Results of the MS homology search for the protein Spot 5II.

<p>The top 10 matches from the search are displayed. Following parameters were used for search: Taxonomy search in <i>Trichomonas vaginalis</i>; Database searched: UniProtKB.2011.01.11; Mass Tolerance: 0.5 Da; Digest Used: No enzyme; Max. # Missed Cleavages: 1; Min matches: 1; Score matrix: BLOSUM62; List of Peptide Sequences: 1 VNVVEGDEADLAT [Q|K] 4, 2 NYW [I|L]VR 1 (number left to the peptide sequence is used as the name of the peptide, whereas the number right to the peptide sequence refers to the maximal number of allowed mismatches). Mismatched amino acids are shown in bold.</p

Percent identity values between cysteine peptidase genes of <i>T. gallinae</i> and their homologues in <i>T. vaginalis</i>.

<p>Percent identity values between cysteine peptidase genes of <i>T. gallinae</i> and their homologues in <i>T. vaginalis</i>.</p

Demonstration of protease activity in cell-free filtrates from <i>T. gallinae</i> clone 5895-C1/06 by one-dimensional substrate SDS-PAGE.

<p>A) Substrate (0.2% gelatin) 1-D SDS-PAGE (8%) with the low passage (49x) cell-free filtrate, B) conventional 1-D SDS PAGE (8%) with the low passage (49x) cell-free filtrate, C) Substrate (0.2% gelatin) 1-D SDS-PAGE (8%) with the high passage (130x) cell-free filtrate, and D) conventional 1-D SDS PAGE (8%) with the high passage (130x) cell-free filtrate.</p

Influence of peptidase inhibitors on the cytopathogenic effect of the cell-free filtrate.

<p>LMH monolayer was incubated with cell-free filtrate with and without different peptidase inhibitors. (A) Mean lesion scores, (B) cytotoxicity of LMH cells, as assessed by Promega CellTiter 96® aqueous solution at different time points. Cell-free filtrate was obtained after 24 h of incubation of 10⁷ axenically grown protozoa cells obtained from <i>T. gallinae</i> clone 5895-C1/06, passage18. Absorbance values for Promega CellTiter 96® aqueous solution were recorded at 490 nm using ELISA reader.</p