Cows with diverging haplotypes show differences in differential milk cell count, milk parameters and vaginal temperature after S. aureus challenge but not after E. coli challenge.

BACKGROUND: In dairy cattle, mastitis causes high financial losses and impairs animal well-being. Genetic selection is used to breed cows with reduced mastitis susceptibility. Techniques such as milk cell flow cytometry may improve early mastitis diagnosis. In a highly standardized in vivo infection model, 36 half-sib cows were selected for divergent paternal Bos taurus chromosome 18 haplotypes (Q vs. q) and challenged with Escherichia coli for 24 h or Staphylococcus aureus for 96 h, after which the samples were analyzed at 12 h intervals. Vaginal temperature (VT) was recorded every three minutes. The objective of this study was to compare the differential milk cell count (DMCC), milk parameters (fat %, protein %, lactose %, pH) and VT between favorable (Q) and unfavorable (q) haplotype cows using Bayesian models to evaluate their potential as improved early indicators of differential susceptibility to mastitis. RESULTS: After S. aureus challenge, compared to the Q half-sibship cows, the milk of the q cows exhibited higher PMN levels according to the DMCC (24 h, p < 0.001), a higher SCC (24 h, p < 0.01 and 36 h, p < 0.05), large cells (24 h, p < 0.05) and more dead (36 h, p < 0.001) and live cells (24 h, p < 0.01). The protein % was greater in Q milk than in q milk at 0 h (p = 0.025). In the S. aureus group, Q cows had a greater protein % (60 h, p = 0.048) and fat % (84 h, p = 0.022) than q cows. Initially, the greater VT of S. aureus-challenged q cows (0 and 12-24 h, p < 0.05) reversed to a lower VT in q cows than in Q cows (48-60 h, p < 0.05). Additionally, the following findings emphasized the validity of the model: in the S. aureus group all DMCC subpopulations (24 h-96 h, p < 0.001) and in the E. coli group nearly all DMCC subpopulations (12 h-24 h, p < 0.001) were higher in challenged quarters than in unchallenged quarters. The lactose % was lower in the milk samples of E. coli-challenged quarters than in those of S. aureus-challenged quarters (24 h, p < 0.001). Between 12 and 18 h, the VT was greater in cows challenged with E. coli than in those challenged with S. aureus (3-h interval approach, p < 0.001). CONCLUSION: This in vivo infection model confirmed specific differences between Q and q cows with respect to the DMCC, milk component analysis results and VT results after S. aureus inoculation but not after E. coli challenge. However, compared with conventional milk cell analysis monitoring, e.g., the global SCC, the DMCC analysis did not provide refined phenotyping of the pathogen response.

Flow cytometric detection and identification of different leukocyte subpopulations in stallion semen.

To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups, 27.85 ± 5.7% of events were positive for CD4, CD8, CD21 or CD172a, while in native semen samples, leukocytes were scarce (0.114 ± 0.134%). The most abundant leukocyte subpopulation in semen was of lymphoid origin (CD4-positive cells [0.015 ± 0.02%]), whereas CD21-positive cells (B cells; 0.001 ± 0.001%) were virtually absent in ejaculates of fertile stallions. This presented flow cytometric assay for the detection and identification of different leukocyte population in equine antioxidant-treated ejaculates can be used as an additional tool for spermatological examination in stallions.

Immune cell composition of the bronchoalveolar lavage fluid in healthy and respiratory diseased dromedary camels.

BACKGROUND: Respiratory diseases are among the most common and expensive to treat diseases in camels with a great economic impact on camel health, welfare, and production. Bronchoalveolar lavage fluid (BALF) has been proven as a valuable sample for investigating the leukocyte populations in the respiratory tract of several species. In the present study, fluorescent antibody labeling and flow cytometry were used to study the immune cell composition of BALF in dromedary camels. Animals with clinical respiratory diseases (n = seven) were compared with apparently healthy animals (n = 10). In addition, blood leukocytes from the same animals were stained in parallel with the same antibodies and analyzed by flow cytometry. RESULTS: Camel BALF macrophages, granulocytes, monocytes, and lymphocytes were identified based on their forward and side scatter properties. The expression pattern of the cell markers CD172a, CD14, CD163, and MHCII molecules on BALF cells indicates a similar phenotype for camel, bovine, and porcine BALF myeloid cells. The comparison between camels with respiratory disease and healthy camels regarding cellular composition in their BALF revealed a higher total cell count, a higher fraction of granulocytes, and a lower fraction of macrophages in diseased than healthy camels. Within the lymphocyte population, the percentages of helper T cells and B cells were also higher in diseased than healthy camels. The elevated expression of the activation marker CD11a on helper T cells of diseased camels is an indication of the expansion of helper T cells population due to infection and exposure to respiratory pathogens. The higher abundance of MHCII molecules on BALF macrophages from diseased camels indicates a polarization toward an inflammatory macrophage phenotype (M1) in respiratory diseased camels. No significant differences were observed in the systemic leukogram between healthy and diseased animals. CONCLUSIONS: Collectively, the current study represents the first report on flow cytometric analysis of immune cell composition of bronchoalveolar lavage fluid (BALF) in dromedary camels.

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes.

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

Triple (GGTA1, CMAH, B2M) modified pigs expressing an SLA class Ilow phenotype-Effects on immune status and susceptibility to human immune responses.

Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9-mediated deletion of ß2 -microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules. Residual SLA class I was functionally inert, because no proper differentiation of the CD8+ T cell subset was observed in B2Mlow pigs. Cells from B2Mlow pigs were less capable in triggering proliferation of human peripheral blood mononuclear cells in vitro, which was mainly due to the nonresponsiveness of CD8+ T cells. Nevertheless, cytotoxic effector cells developing from unaffected cell populations (eg, CD4+ T cells, natural killer cells) lysed targets from both SLA class I+ wildtype and SLA class Ilow pigs with similar efficiency. These data indicate that the absence of SLA class I is an effective approach to prevent the activation of human CD8+ T cells during the induction phase of an anti-xenograft response. However, cytotoxic activity of cells during the effector phase cannot be controlled by this approach.

Blood leukocyte composition and function in periparturient ewes kept on different dietary magnesium supply.

BACKGROUND: Transition period (TP) is characterised by physiological and metabolic changes contributing to immunodysregulation. Since knowledge about this period in sheep is scarce, we analysed changes in selected immune variables during the TP in ewes and whether dietary magnesium (Mg) supplementation could modulate these immune variables. Pregnant ewes (2nd and 3rd lactation) were divided into a control group (CONT, n = 9) and a Mg group (MAG, n = 10) supplemented with Mg oxide resulting in a daily Mg intake of approximately 0.30 and 0.38% (MAG) of dry matter during ante- (a.p.) and post-partum (p.p.) periods, respectively. Blood samples were collected between days (d) 30 a.p. and d 30 p.p.. Whole blood neutrophil phagocytic activity, monocyte subset (classical cM, intermediate intM, non-classical ncM) composition and the proliferative capacity of lymphocytes were determined flow cytometrically. At d 14 a.p., all ewes were vaccinated against Mycobacterium avium subsp. paratuberculosis (MAP). RESULTS: Both groups showed a sharp increase in the total leukocyte counts (TLC) and neutrophil counts (P < 0.0001), at d 1 p.p., while, monocytes and their subpopulations displayed the highest values at d 30 p.p. (P ≤ 0.05). At d 1 p.p. the neutrophil phagocytic activity was higher (P < 0.05) in MAG ewes. Throughout the TP, the proliferative response of CD4+ cells was significantly higher in the MAG group (P < 0.05). Ewes in both groups responded with an increase in the TLC, neutrophil numbers (P ≤ 0.05) and ncM (P < 0.001) 24 h post vaccination, whereas monocytes and cM dropped in numbers (P ≤ 0.05). Numbers of intM only increased in MAG ewes (P < 0.05), whereas lymphocyte numbers decreased (P < 0.01). Mg supplementation did not affect the significant increase in MAP-specific antibodies at d 7 and 21 post vaccination. Total Mg and Ca serum levels did not show any differences between the two groups. CONCLUSION: Whereas TP-associated fluctuations in blood leukocyte numbers are not influenced by Mg supplementation, neutrophil phagocytic activity, the proliferative capacity of CD4+ cells and the cellular response within 24 h after a vaccination are subject to modulation.

Dromedary camel CD14high MHCIIhigh monocytes display inflammatory properties and are reduced in newborn camel calves.

BACKGROUND: In human and different animal species, blood monocytes are classified based on their expression pattern of different monocytic markers into phenotypically and functionally different subsets. In the current study, we used flow cytometry and monoclonal antibodies to CD172a, CD14, CD163 and MHCII to identify monocyte subsets in peripheral blood of dromedary camels. RESULTS: Based on CD14, CD163 and MHCII expression, camel CD172a + monocytes were divided into three subsets: The major subpopulation of camel monocytes (mo-I) showed high expression of CD14 and CD163, but low expression of MHCII. A second subset of monocytes (mo-II) expressed highly all three markers, CD14, CD163 and MHCII. A third monocyte subset (mo-III) displayed low expression of CD14 and CD163 with high MHCII expression. While the two MHCIIhigh subsets (mo-II and mo-III) showed higher expression of CD11a in comparison to the MHCIIlow subset (mo-I), CD18 and CD11b were highest expressed on the two CD14high subsets (mo-I and mo-II). Bacterial stimulation of camel leukocytes identified mo-II cells as an antimicrobial monocyte subset with the highest phagocytic and ROS production capacity. The comparison of monocyte counts and phenotype between newborn calves and adult camels revealed significantly reduced numbers of mo-II cells in newborn animals. Monocytes of newborns expressed significantly more CD172a and CD163 molecules but less CD14 and MHCII molecules than monocytes of adult camels. CONCLUSIONS: Camel monocyte subsets, mo-I, mo-II and mo-III are counterparts of bovine classical, intermediate and non-classical monocytes respectively. The distribution of camel monocyte subsets is influenced by age.

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue.

BACKGROUND: In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. RESULTS: Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins (CSN1S1, CSN1S2, CSN2 and CSN3) and whey proteins (LALBA and PAEP) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli (E. coli) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challenge-associated biological signaling pathway patterns. CONCLUSIONS: The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity.

Dietary Fatty Acids Affect Red Blood Cell Membrane Composition and Red Blood Cell ATP Release in Dairy Cows.

Diets of dairy cows are often based on maize silage (MS), delivering lower amounts of n-3 fatty acids (FA) compared to grass silage-based diets. The fatty acid composition of the cell membrane can affect the cell function. We evaluated the effects of an MS-based diet on bovine red blood cell (RBC) membrane FA composition and dietary effects on controlled ATP release of RBC. In trial 1, German Holstein cows were fed an MS-based total mixed ration for 24 weeks. The FA composition of RBC membranes from repeatedly taken blood samples was analysed in addition to the abundance of the RBC membrane protein flotillin-1, which is involved in, for example, cell signalling. In trial 2, four rumen fistulated MS-fed cows were abomasally infused in a 4 × 4 Latin square model with three successively increasing lipid dosages (coconut oil, linseed-safflower oil mix (EFA; rich in n-3 FA), Lutalin®, providing conjugated linoleic acids (CLA) or the combination of the supplements, EFA + CLA) for six weeks, followed by a three-week washout period. In trial 2, we analysed RBC ATP release, flotillin-1, and the membrane protein abundance of pannexin-1, which is involved in ATP release as the last part of a signalling cascade. In trial 1, the total amount of n-3 FA in RBC membranes decreased and the flotillin-1 abundance increased over time. In trial 2, the RBC n-3 FA amount was higher after the six-week infusion period of EFA or EFA + CLA. Furthermore, depending on the dosage of FA, the ATP release from RBC increased. The abundance of flotillin-1 and pannexin-1 was not affected in trial 2. It is concluded that changes of the membrane FA composition influence the RBC function, leading to altered ATP release from intact bovine RBC.

Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis.

Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies.

Counts of bovine monocyte subsets prior to calving are predictive for postpartum occurrence of mastitis and metritis.

The heightened susceptibility to infectious diseases in postpartum dairy cows is often attributed to immune dysfunction associated with the transition period. However, the cell populations involved in this immune dysfunction and the dynamics between those populations are not well defined. Monocytes play a crucial role in governing initial immune response in bacterial infections. Bovine monocytes are subdivided in classical (CD14+/CD16-), intermediate (CD14+/CD16+) and non-classical monocytes (CD14-/CD16+) with distinct phenotypic and functional differences. This study investigated the relationship of monocyte subsets counts in blood at 42 and 14 days prior to expected calving date to occurrence of metritis and mastitis within 2 weeks postpartum. In the enrolled prospective cohort of 27 German Holstein cows, housed at the Institute of Animal Nutrition of the Friedrich-Loeffler-Institute Braunschweig, Germany, n = 13 developed metritis and/or mastitis postpartum. A multivariable logistic regression was used to analyze the relationship between prepartum cell counts of monocyte subsets and neutrophils with postpartum disease. Our model revealed that higher counts of the two CD14+ monocyte subsets were predictive of disease. In contrast, higher numbers of the CD14- monocyte subset were negatively associated with disease. Interestingly, the neutrophil count, a common hallmark for inflammatory response, was not associated with the outcome variable at either time point. The results indicate that the number and composition of monocyte subsets before calving are related to the susceptibility to infectious disease within 2 weeks postpartum. Furthermore the oppositional effect of CD14+ and CD14- subsets strengthens the hypothesis that these subsets have different functional roles in the inflammatory response in dairy cows.

Comparison of the pathogen species-specific immune response in udder derived cell types and their models.

The outcome of an udder infection (mastitis) largely depends on the species of the invading pathogen. Gram-negative pathogens, such as Escherichia coli often elicit acute clinical mastitis while Gram-positive pathogens, such as Staphylococcus aureus tend to cause milder subclinical inflammations. It is unclear which type of the immune competent cells residing in the udder governs the pathogen species-specific physiology of mastitis and which established cell lines might provide suitable models. We therefore profiled the pathogen species-specific immune response of different cell types derived from udder and blood. Primary cultures of bovine mammary epithelial cells (pbMEC), mammary derived fibroblasts (pbMFC), and bovine monocyte-derived macrophages (boMdM) were challenged with heat-killed E. coli, S. aureus and S. uberis mastitis pathogens and their immune response was scaled against the response of established models for MEC (bovine MAC-T) and macrophages (murine RAW 264.7). Only E. coli provoked a full scale immune reaction in pbMEC, fibroblasts and MAC-T cells, as indicated by induced cytokine and chemokine expression and NF-κB activation. Weak reactions were induced by S. aureus and none by S. uberis challenges. In contrast, both models for macrophages (boMdM and RAW 264.7) reacted strongly against all the three pathogens accompanied by strong activation of NF-κB factors. Hence, the established cell models MAC-T and RAW 264.7 properly reflected key aspects of the pathogen species-specific immune response of the respective parental cell type. Our data imply that the pathogen species-specific physiology of mastitis likely relates to the respective response of MEC rather to that of professional immune cells.

Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma.

BACKGROUND: Equine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow-cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p-value < 0.05 was considered significant for all statistical tests applied. RESULTS: In all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination. CONCLUSIONS: This is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect.

Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma.

BACKGROUND: Equine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow-cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p-value < 0.05 was considered significant for all statistical tests applied. RESULTS: In all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination. CONCLUSIONS: This is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect.

Genomic characterisation of an endometrial pathogenic Escherichia coli strain reveals the acquisition of genetic elements associated with extra-intestinal pathogenicity.

BACKGROUND: Strains of Escherichia coli cause a wide variety of intestinal and extra-intestinal diseases in both humans and animals, and are also often found in healthy individuals or the environment. Broadly, a strong phylogenetic relationship exists that distinguishes most E. coli causing intestinal disease from those that cause extra-intestinal disease, however, isolates within a recently described subclass of Extra-Intestinal Pathogenic E. coli (ExPEC), termed endometrial pathogenic E. coli, tend to be phylogenetically distant from the vast majority of characterised ExPECs, and more closely related to human intestinal pathogens. In this work, we investigate the genetic basis for ExPEC infection in the prototypic endometrial pathogenic E. coli strain MS499. RESULTS: By investigating the genome of MS499 in comparison with a range of other E. coli sequences, we have discovered that this bacterium has acquired substantial lengths of DNA which encode factors more usually associated with ExPECs and less frequently found in the phylogroup relatives of MS499. Many of these acquired factors, including several iron acquisition systems and a virulence plasmid similar to that found in several ExPECs such as APEC O1 and the neonatal meningitis E. coli S88, play characterised roles in a variety of typical ExPEC infections and appear to have been acquired recently by the evolutionary lineage leading to MS499. CONCLUSIONS: Taking advantage of the phylogenetic relationship we describe between MS499 and several other closely related E. coli isolates from across the globe, we propose a step-wise evolution of a novel clade of sequence type 453 ExPECs within phylogroup B1, involving the recruitment of ExPEC virulence factors into the genome of an ancestrally non-extraintestinal E. coli, which has repurposed this lineage with the capacity to cause extraintestinal disease. These data reveal the genetic components which may be involved in this phenotype switching, and argue that horizontal gene exchange may be a key factor in the emergence of novel lineages of ExPECs.

Differential endometrial cell sensitivity to a cholesterol-dependent cytolysin links Trueperella pyogenes to uterine disease in cattle.

Purulent disease of the uterus develops in 40% of dairy cows after parturition, when the epithelium of the endometrium is disrupted to expose the underlying stroma to bacteria. The severity of endometrial pathology is associated with isolation of Trueperella pyogenes. In the present study, T. pyogenes alone caused uterine disease when infused into the uterus of cattle where the endometrial epithelium was disrupted. The bacterium secretes a cholesterol-dependent cytolysin, pyolysin (PLO), and the plo gene was identical and the plo gene promoter was highly similar amongst 12 clinical isolates of T. pyogenes. Bacteria-free filtrates of the T. pyogenes cultures caused hemolysis and endometrial cytolysis, and PLO was the main cytolytic agent, because addition of anti-PLO antibody prevented cytolysis. Similarly, a plo-deletion T. pyogenes mutant did not cause hemolysis or endometrial cytolysis. Endometrial stromal cells were notably more sensitive to PLO-mediated cytolysis than epithelial or immune cells. Stromal cells also contained more cholesterol than epithelial cells, and reducing stromal cell cholesterol content using cyclodextrins protected against PLO. Although T. pyogenes or plo-deletion T. pyogenes stimulated accumulation of inflammatory mediators, such as IL-1beta, IL-6, and IL-8, from endometrium, PLO did not stimulate inflammatory responses by endometrial or hematopoietic cells, or in vitro organ cultures of endometrium. The marked sensitivity of stromal cells to PLO-mediated cytolysis provides an explanation for how T. pyogenes acts as an opportunistic pathogen to cause pathology of the endometrium once the protective epithelium is lost after parturition.

Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis.

BACKGROUND: Molecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples. RESULTS: In lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE. CONCLUSIONS: Real-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells.

Supplementation of Foals with a Saccharomyces cerevisiae Fermentation Product Alters the Early Response to Vaccination.

Feed supplements supporting animal welfare and performance are becoming increasingly important. Immunomodulatory effects of such products have been observed in many species. The aim of this study was to analyze whether food supplementation with a Saccharomyces cerevisiae fermentation product (SCFP) affects the occurrence of foal diarrhea in early life, and whether the SCFP feeding has an impact on the immediate response to a parenteral vaccination at the age of 6-9 months. Eleven foals received the SCFP (OLI) and eleven foals were fed a placebo (PLA) for 29 days. Growth, diarrhea, and diarrhea severity were observed until day 30. After weaning, at the age of 6-9 months, foals were vaccinated parenterally against influenza and tetanus. The supplementation had no statistically significant effect on diarrhea duration and severity. On the day of vaccination, PLA and OLI foals did not differ significantly regarding numbers of circulating blood leukocyte subsets. However, the response to vaccination differed significantly between OLI and PLA foals. In OLI foals, the numbers of the major leukocyte fractions (granulocytes, lymphocytes, monocytes, CD4+ T cells, CD8+ T cells, CD21+ B cells, and MHC-II+/CD21- cells) increased significantly 24 h after vaccination but remained unchanged in PLA foals. The observed results suggest that early life supplementation with an SCFP may affect the early immune response to an initial vaccination.

Escherichia coli- and Staphylococcus aureus-induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters.

BACKGROUND: The most important disease of dairy cattle is mastitis, caused by the infection of the mammary gland by various micro-organisms. Although the transcriptional response of bovine mammary gland cells to in vitro infection has been studied, the interplay and consequences of these responses in the in vivo environment of the mammary gland are less clear. Previously mammary gland quarters were considered to be unaffected by events occurring in neighbouring quarters. More recently infection of individual quarters with mastitis causing pathogens, especially Escherichia coli, has been shown to influence the physiology of neighbouring uninfected quarters. Therefore, the transcriptional responses of uninfected mammary gland quarters adjacent to quarters infected with two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were compared. RESULTS: The bacteriologically sterile, within-animal control quarters exhibited a transcriptional response to the infection of neighbouring quarters. The greatest response was associated with E. coli infection, while a weaker, yet significant, response occurred during S. aureus infection. The transcriptional responses of these uninfected quarters included the enhanced expression of many genes previously associated with mammary gland infections. Comparison of the transcriptional response of uninfected quarters to S. aureus and E. coli infection identified 187 differentially expressed genes, which were particularly associated with cellular responses, e.g. response to stress. The most affected network identified by Ingenuity Pathway analysis has the immunosuppressor transforming growth factor beta 1 (TGFB1) at its hub and largely consists of genes more highly expressed in control quarters from S. aureus infected cows. CONCLUSIONS: Uninfected mammary gland quarters reacted to the infection of neighbouring quarters and the responses were dependent on pathogen type. Therefore, bovine udder quarters exhibit interdependence and should not be considered as separate functional entities. This suggests that mastitis pathogens not only interact directly with host mammary cells, but also influence discrete sites some distance away, which will affect their response to the subsequent spread of the infection. Understanding the underlying mechanisms may provide further clues for ways to control mammary gland infections. These results also have implications for the design of experimental studies investigating immune regulatory mechanisms in the bovine mammary gland.

Polarised bovine endometrial epithelial cells vectorially secrete prostaglandins and chemotactic factors under physiological and pathological conditions.

Epithelial cells of the endometrium secrete prostaglandins to regulate the bovine oestrous cycle and form a functional barrier to microbes. However, bacterial infection of the endometrium commonly causes infertility in dairy cattle by disrupting endometrial physiology. Epithelial cell cultures are used to study the mechanisms of physiology and pathology, but 2D cultures may not reflect the 3D complexity of the epithelium. In this study, a polarised epithelial cell transwell culture was developed, using transepithelial resistance (TER), to monitor epithelial integrity. Polarised epithelial cells were treated with oxytocin and arachidonic acid to test physiological function and with lipopolysaccharide (LPS) to mimic bacterial infection. Supernatants were analysed for prostaglandin E(2) (PGE), prostaglandin F(2)(α), the chemokine interleukin-8 (IL8) and the ability of supernatants to induce neutrophil migration. Confluent epithelial cells established polarity when TER was >1800  Ω cm(2) and predominantly released prostaglandins basolaterally. In contrast, IL8 from epithelial cells accumulated apically and the supernatants were highly chemotactic for neutrophils. The striking exception was when the epithelial cells were treated with LPS in the apical or basolateral compartment independently, which led to the release of IL8 towards the treated compartment. Although stromal cells also accumulated PGE and IL8 in response to treatment, co-culture of stromal cells in the well below polarised epithelial cells did not influence cellular responses. In conclusion, polarised endometrial epithelial cells vectorially released prostaglandins and chemokines to reflect their respective mechanistic roles in physiology and pathology.