- Email: [email protected]
26. Induction of clinically unobservable inflammations by E. coli endotoxin
174
Poster Presentation Abstracts
acid sequence of a peptidic fragment obtained from corpora amylacea gave clear evidence of the occurrence of p-casein. Further determination by western-blot showed that the main milk proteins (caseins and whey proteins) are implicated in the development of corpora amylacea. But, the comparison between the amino acid composition of amyloid bodies and those of the main milk proteins revealed that these structures were especially rich in serine and poor in tyrosine and phenylalanine. Thus, corpora amylacea were composed of more than milk proteins. It is likely that the composition of amyloid bodies is much more complex than first reported. 25. A comparison of symptoms in subclinical and clinical mastitis cases in cows. A. Berman*, E. Shoshani and B. Hanochi. Department of Animal Science, Faculty of Agriculture, Hebrew University, Rehovot 76100, Israel. Computerised milking systems, like the Afimilk system, detect combined reductions in yield and in electrical resistance of composite milk. Such events were associated with high SCC (somatic cell count) and NAGase (N-acetyl-/?-D-glucosaminidase), occurred on the background of infection, and were followed by marked milk losses. Only 20 to 30% of such combined reductions evolved into clinical mastitis. This study aimed to characterise such types of mastitis inflammation cases that were not hitherto reported. It examined 16 such spontaneous cases, 11 clinical and 5 subclinical. Cases were considered clinical if 2 of 3 symptoms (clots, gland swollen and gland redness) were observed on the day of detection of the combined reductions of milk yield and electrical resistance. Composite milk samples taken on day of detection and on days 2, 3, 5, 8 and 12 thereafter served for bacteriological culture, NAGase, fat, total protein, SCC, casein, albumin, and pH determination. Pathogens detected varied widely between cases and between days within case. There were no distinct differences between clinical and subclinical cases in types of pathogens. All cases were linked with rises in SCC, and all clinical and most subclinical cases with rises in NAGase. In the clinical cases, SCC and NAGase were significantly higher than in the subclinical cases (2.6 X lo6 vs. 0.7 X 106; 37 vs. 90, respectively); total protein was also 0.15% units higher, owing to a rise in albumin content. Milk fat and lactose were reduced by 0.1 to 0.2% units in the clinical cases. Trends over time were similar in clinical and subclinical cases: changes were most marked on day of detection, to gradually fade out, unless a second rise in inflammation occurred. A second rise in inflammation occurred in three of the cases, one after a clinical inflammation and the other two cases after events that remained subclinical. The study indicates that subclinical and clinical inflammations were similar in parameters measured, and differed in the magnitude of the changes. The results also indicate that combined changes in milk yield and electrical resistance might detect rises in inflammation that would, otherwise, remain unobserved. 26. Induction of clinically unobservable inflammations by E. coli endotoxin. E. Shoshani’ * , G. Leitner*, A. Saran* and A. Berman’. ‘Dept. An’imal Science, Fat. Agric., Hebrew Univ. Rehovot; *Mastitis Reference Dept., Kimron Vet. Inst., Bet Dagan, Israel. Mastitis is an infection that may erupt into clinical form. Recently, recurrent inflammations, intermediate between chronic and clinical mastitis states, have been observed. Experimental models have been developed for study of chronic infection and of clinical mastitis, but no such model is known for mastitis states that are intermediate between chronic infection and clinically observable states. This reports a tentative experimental model for the study of mastitis inflammations that do not evolve to a clinically observable form. After milking, 0.5 pg of E. coli endotoxin were administered into two diagonal quarters chronically infected by S. aureus in each of three Holstein cows. Two quarters of another cow, free of infection, served as control as well as a cow in which a spontaneous inflammation occurred. Clinical examination was carried out every 2 h up to 8 h past-treatment (PT). Quarter samples were taken from -72 to 144 h PT, and examined for Cl, Na, K, NAGase (N-acetyl-13-D-glucosaminidase), albumin, somatic cell count, differential leukocyte count and phagocytosis (PH). No pyrogenic or clinical symptoms were observed. Leukocytosis appeared in treated quarters 2 h PT and peaked between 4 and 24 h PT. NAGase, Na, Cl, and albumin increased, while K decreased after treatment in a pattern similar to that of the leukocytes. During peak leukocytosis, part of the neutrophils and macrophages
Poster Presentation Abstracts
175
were unlabeled by monoclonal antibodies for these mature cells. Total lymphocytes increased moderately for 72 bearing BOCD4+. PH was h PT, mostly T-lymphocytes, bearing BOCD8+, followed by T-lymphocytes depressed until 72 h PT. A spontaneous eruption of an intermediate state of mastitis in a quarter chronically infected by S. aureu~ was similar to that in the experimentally induced intermediate state. These indicate that an intermediate, subclinical inflammation may be produced by a small dose of E. coli endotoxin. AS the same endotoxin is used in higher doses, as a model for clinical mastitis, the results suggest that clinical and intermediate mastitis inflammations might represent states that differ quantitatively but are similar qualitatively. 27. Characterisation of the bovine STAT5/MGF transcription factor, a mediator of prolactin. H.-M. Seyfert* and P. Schroder. Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany. Our objective was to characterise via sequencing the cDNA of the bovine ‘mammary gland factor’ as necessary first step to isolate the encoding gene. Interest in the characterisation of high (STAT~/MGF) performance variants of this gene stems from the fact that the STATS/MGF factor transduces the prolactin signal, inducing casein-synthesis at the beginning of lactation. Hence, performance variants of this gene might influence the quantitative yields of casein synthesis. The complete sequence of the bovine factor was compiled (i) from a cDNA-fragment, covering about 1200 bp from the 5’-end of the molecule, which was isolated from our mammary gland specific cDNA library, while (ii) the 3’-end was established using reverse Polymerase Chain Reaction techniques (PCR), followed by cloning of the products. Primers for PCR-techniques were derived from a previously reported ovine sequence of the respective factor. The bovine STATS/MGF encodes a protein comprising 794 amino acids (aa), with a phosphorylable Tyr-residue at position 694, just as its ovine and murine counterparts. The homology between the bovine and murine factor STATSA is higher at the protein level (95.8% over 794 aa residues) than at the nucleotide level (87.6% over 2578 bp overlap). Our bovine factor conforms to the murine STATSA homologue rather than to STATSB. The reported ovine STAT5 sequence contains a substantial insertion ( > 100 bp) in the 5’-untranslated region. This represents an extra exon, since the break-points of cDNA homology are matched by splice-boundaries of the bovine gene. 28. In vitro effect of /.$hydroxybutyric acid on the chemiluminescence of bovine blood neutrophils. D. Hoeben * and C. Burvenich. University of Ghent, Merelbeke, Belgium. In high yielding cows shortly after parturition, the blood level of butyric acid increases, indicating a negative energy balance. In the same period, an increased incidence of metabolic and infectious diseases has been observed. Several epidemiological studies showed a link among negative energy balance, elevated occurrence of diseases such as ketosis, E. coli mastitis, met&is and an impaired function of neutrophils. The purpose of this study was to evaluate the in vitro effect of normal (0.01 to 1 mM) and ketotic (1 to 5 mM) doses of butyric acid on the respiratory burst activity of bovine polymorphonuclear leukocytes (PMNL) isolated from blood. The reactive oxygen species generating capacity was measured by means of the luminol-enhanced, PMA (phorbol 12-myristate 13-acetate)-induced chemiluminescence assay (CL). The results showed a significant inhibitory effect of ketotic concentrations of butyric acid on CL of isolated PMNL with a maximal inhibition of 15% (P < 0.001). In a cell-free assay consisting of sonicated cells and hydrogen peroxide, no changes in activity could be observed. The activity of the myeloperoxidase was not significantly affected as shown by the ortho-dianisidine oxidation assay. Also, the production of superoxide radicals measured by the cytochrome c reduction assay was not altered by different doses of butyric acid. Butyric acid had no scavenging effect on hypochlorite as shown by a CL assay using hypochlorite and luminol. The reason for the inhibitory effect on CL may be a decreased production of hydrogen peroxide. Indeed, luminol-enhanced CL gives an idea of the production of this oxygen species. This could not be confirmed in the other assays mentioned above because hydrogen peroxide was added externally in these assays. In conclusion, because of this inhibitory effect on the respiratory burst activity of PMNL, the elevated blood level of butyric acid after parturition in high yielding cows may be, in part, responsible for the higher susceptibility to local and systemic infections during the postpartum period and during subclinical and clinical ketosis.
Poster Presentation Abstracts
acid sequence of a peptidic fragment obtained from corpora amylacea gave clear evidence of the occurrence of p-casein. Further determination by western-blot showed that the main milk proteins (caseins and whey proteins) are implicated in the development of corpora amylacea. But, the comparison between the amino acid composition of amyloid bodies and those of the main milk proteins revealed that these structures were especially rich in serine and poor in tyrosine and phenylalanine. Thus, corpora amylacea were composed of more than milk proteins. It is likely that the composition of amyloid bodies is much more complex than first reported. 25. A comparison of symptoms in subclinical and clinical mastitis cases in cows. A. Berman*, E. Shoshani and B. Hanochi. Department of Animal Science, Faculty of Agriculture, Hebrew University, Rehovot 76100, Israel. Computerised milking systems, like the Afimilk system, detect combined reductions in yield and in electrical resistance of composite milk. Such events were associated with high SCC (somatic cell count) and NAGase (N-acetyl-/?-D-glucosaminidase), occurred on the background of infection, and were followed by marked milk losses. Only 20 to 30% of such combined reductions evolved into clinical mastitis. This study aimed to characterise such types of mastitis inflammation cases that were not hitherto reported. It examined 16 such spontaneous cases, 11 clinical and 5 subclinical. Cases were considered clinical if 2 of 3 symptoms (clots, gland swollen and gland redness) were observed on the day of detection of the combined reductions of milk yield and electrical resistance. Composite milk samples taken on day of detection and on days 2, 3, 5, 8 and 12 thereafter served for bacteriological culture, NAGase, fat, total protein, SCC, casein, albumin, and pH determination. Pathogens detected varied widely between cases and between days within case. There were no distinct differences between clinical and subclinical cases in types of pathogens. All cases were linked with rises in SCC, and all clinical and most subclinical cases with rises in NAGase. In the clinical cases, SCC and NAGase were significantly higher than in the subclinical cases (2.6 X lo6 vs. 0.7 X 106; 37 vs. 90, respectively); total protein was also 0.15% units higher, owing to a rise in albumin content. Milk fat and lactose were reduced by 0.1 to 0.2% units in the clinical cases. Trends over time were similar in clinical and subclinical cases: changes were most marked on day of detection, to gradually fade out, unless a second rise in inflammation occurred. A second rise in inflammation occurred in three of the cases, one after a clinical inflammation and the other two cases after events that remained subclinical. The study indicates that subclinical and clinical inflammations were similar in parameters measured, and differed in the magnitude of the changes. The results also indicate that combined changes in milk yield and electrical resistance might detect rises in inflammation that would, otherwise, remain unobserved. 26. Induction of clinically unobservable inflammations by E. coli endotoxin. E. Shoshani’ * , G. Leitner*, A. Saran* and A. Berman’. ‘Dept. An’imal Science, Fat. Agric., Hebrew Univ. Rehovot; *Mastitis Reference Dept., Kimron Vet. Inst., Bet Dagan, Israel. Mastitis is an infection that may erupt into clinical form. Recently, recurrent inflammations, intermediate between chronic and clinical mastitis states, have been observed. Experimental models have been developed for study of chronic infection and of clinical mastitis, but no such model is known for mastitis states that are intermediate between chronic infection and clinically observable states. This reports a tentative experimental model for the study of mastitis inflammations that do not evolve to a clinically observable form. After milking, 0.5 pg of E. coli endotoxin were administered into two diagonal quarters chronically infected by S. aureus in each of three Holstein cows. Two quarters of another cow, free of infection, served as control as well as a cow in which a spontaneous inflammation occurred. Clinical examination was carried out every 2 h up to 8 h past-treatment (PT). Quarter samples were taken from -72 to 144 h PT, and examined for Cl, Na, K, NAGase (N-acetyl-13-D-glucosaminidase), albumin, somatic cell count, differential leukocyte count and phagocytosis (PH). No pyrogenic or clinical symptoms were observed. Leukocytosis appeared in treated quarters 2 h PT and peaked between 4 and 24 h PT. NAGase, Na, Cl, and albumin increased, while K decreased after treatment in a pattern similar to that of the leukocytes. During peak leukocytosis, part of the neutrophils and macrophages
Poster Presentation Abstracts
175
were unlabeled by monoclonal antibodies for these mature cells. Total lymphocytes increased moderately for 72 bearing BOCD4+. PH was h PT, mostly T-lymphocytes, bearing BOCD8+, followed by T-lymphocytes depressed until 72 h PT. A spontaneous eruption of an intermediate state of mastitis in a quarter chronically infected by S. aureu~ was similar to that in the experimentally induced intermediate state. These indicate that an intermediate, subclinical inflammation may be produced by a small dose of E. coli endotoxin. AS the same endotoxin is used in higher doses, as a model for clinical mastitis, the results suggest that clinical and intermediate mastitis inflammations might represent states that differ quantitatively but are similar qualitatively. 27. Characterisation of the bovine STAT5/MGF transcription factor, a mediator of prolactin. H.-M. Seyfert* and P. Schroder. Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany. Our objective was to characterise via sequencing the cDNA of the bovine ‘mammary gland factor’ as necessary first step to isolate the encoding gene. Interest in the characterisation of high (STAT~/MGF) performance variants of this gene stems from the fact that the STATS/MGF factor transduces the prolactin signal, inducing casein-synthesis at the beginning of lactation. Hence, performance variants of this gene might influence the quantitative yields of casein synthesis. The complete sequence of the bovine factor was compiled (i) from a cDNA-fragment, covering about 1200 bp from the 5’-end of the molecule, which was isolated from our mammary gland specific cDNA library, while (ii) the 3’-end was established using reverse Polymerase Chain Reaction techniques (PCR), followed by cloning of the products. Primers for PCR-techniques were derived from a previously reported ovine sequence of the respective factor. The bovine STATS/MGF encodes a protein comprising 794 amino acids (aa), with a phosphorylable Tyr-residue at position 694, just as its ovine and murine counterparts. The homology between the bovine and murine factor STATSA is higher at the protein level (95.8% over 794 aa residues) than at the nucleotide level (87.6% over 2578 bp overlap). Our bovine factor conforms to the murine STATSA homologue rather than to STATSB. The reported ovine STAT5 sequence contains a substantial insertion ( > 100 bp) in the 5’-untranslated region. This represents an extra exon, since the break-points of cDNA homology are matched by splice-boundaries of the bovine gene. 28. In vitro effect of /.$hydroxybutyric acid on the chemiluminescence of bovine blood neutrophils. D. Hoeben * and C. Burvenich. University of Ghent, Merelbeke, Belgium. In high yielding cows shortly after parturition, the blood level of butyric acid increases, indicating a negative energy balance. In the same period, an increased incidence of metabolic and infectious diseases has been observed. Several epidemiological studies showed a link among negative energy balance, elevated occurrence of diseases such as ketosis, E. coli mastitis, met&is and an impaired function of neutrophils. The purpose of this study was to evaluate the in vitro effect of normal (0.01 to 1 mM) and ketotic (1 to 5 mM) doses of butyric acid on the respiratory burst activity of bovine polymorphonuclear leukocytes (PMNL) isolated from blood. The reactive oxygen species generating capacity was measured by means of the luminol-enhanced, PMA (phorbol 12-myristate 13-acetate)-induced chemiluminescence assay (CL). The results showed a significant inhibitory effect of ketotic concentrations of butyric acid on CL of isolated PMNL with a maximal inhibition of 15% (P < 0.001). In a cell-free assay consisting of sonicated cells and hydrogen peroxide, no changes in activity could be observed. The activity of the myeloperoxidase was not significantly affected as shown by the ortho-dianisidine oxidation assay. Also, the production of superoxide radicals measured by the cytochrome c reduction assay was not altered by different doses of butyric acid. Butyric acid had no scavenging effect on hypochlorite as shown by a CL assay using hypochlorite and luminol. The reason for the inhibitory effect on CL may be a decreased production of hydrogen peroxide. Indeed, luminol-enhanced CL gives an idea of the production of this oxygen species. This could not be confirmed in the other assays mentioned above because hydrogen peroxide was added externally in these assays. In conclusion, because of this inhibitory effect on the respiratory burst activity of PMNL, the elevated blood level of butyric acid after parturition in high yielding cows may be, in part, responsible for the higher susceptibility to local and systemic infections during the postpartum period and during subclinical and clinical ketosis.