Serum alkaline phosphatase isoenzyme profiles in phenobarbital-treated epileptic dogs

BACKGROUND: Serum total alkaline phosphatase (AP) activity commonly is high in dogs receiving phenobarbital. Specific isoenzymes responsible for this increase are not well documented. OBJECTIVES: The purposes of this study were 1) to qualitatively and quantitatively describe serum AP isoenzymes in phenobarbital-treated dogs and 2) to monitor changes in serum AP isoenzyme activities associated with phenobarbital treatment over time. METHODS: Serum AP isoenzyme activities were determined in a cross-sectional study of 29 dogs receiving phenobarbital (duration of treatment 2 months to 6.5 years). Additionally, in a prospective study of 23 dogs, serum AP isoenzyme activities were determined before and 3 weeks, 6 months, and 12 months after the start of phenobarbital treatment. Isoenzyme activities were quantitatively determined using wheat germ lectin precipitation and levamisole inhibition, and qualitatively (ie, present or absent) evaluated using cellulose acetate affinity electrophoresis. RESULTS: In phenobarbital-treated dogs with high serum total AP activity in the cross-sectional study, the increase was due predominantly to increased activities of the corticosteroid-induced (C-AP) and liver (L-AP) isoenzymes. Prospectively, serum total AP and L-AP activities were significantly higher at 3 weeks, 6 months, and 12 months after the start of phenobarbital treatment compared with pretreatment values. Serum C-AP and bone isoenzyme (B-AP) activities were significantly higher after 6 and 12 months of treatment. B-AP accounted for only a small amount of the total AP activity. No unusual or previously unidentified AP isoenzymes were identified. CONCLUSIONS: Phenobarbital treatment was associated with increased C-AP and L-AP isoenzyme activities and with a minor increase in B-AP activity. No unique "phenobarbital-induced" isoenzyme was identified. Isoenzyme analysis does not appear to be useful for differentiating between high serum total AP due to phenobarbital therapy and other causes.     

Innate immune response to intramammary infection with Serratia marcescens and Streptococcus uberis

Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1 beta, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1 beta, IL-10, IL-12, IFN-gamma, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens.     

Influence of emulsifiers and fat mixtures on the digestibility and sedimentation of fatty acids in calves

In a digestibility experiment with 4 X 4 calves the animals received 100 g mixture for calves, 50 g dried green fodder and either 656 g dried whole milk and 164 g dried skim milk (VM) or 656 g dried whole milk and 164 g added fat. The added fat consisted of a mixture of tallow and lard in a 1:1 ratio plus 10% emulgator ES 20 (FE) or 8% ES 20 and 2% soybean lecithin (FL) or 25% lard, 25% tallow, 40% rape/sunflower oil with 10% ES 20 (FO). The apparent digestibility of the fat amounted to 91% in group VM and in groups FE/FL/FO to 66/70/67% resp. The composition of fatty acids and the use of an unsuitable charge of dried skim milk are considered to be the causes of the low apparent digestibility. The apparent digestibility of the fatty acids decreased with their growing chain length. The higher digestibility of the unsaturated fatty acids is largely caused by changes due to bacterial activity in these fatty acids in the intestines and by the influence of metabolically changed faecal fat. The combination of the synthetic emulgator with lecithin did not improve fat digestion but diminished the total fat content in the blood.     

Analysis of selective mobilization of L-selectin and Mac-1 reservoirs in bovine neutrophils and eosinophils

Following activation of granulocytes, L-selectin (CD62L) is generally shed from the cellular surface, whereas Mac-1 (CD11b/CD18) expression is well known to increase. However, a number of studies in bovines and humans show that the expression of L-selectin may increase as well. This urged us to examine the possible existence of both L-selectin and Mac-1 reservoirs in bovine neutrophil and eosinophil populations through the use of flow cytometry in combination with an optimized method for cell membrane permeabilization. Augmented L-selectin and Mac-1 expression was detected in both granulocyte populations upon saponin treatment. Confocal microscopic studies indicated that both molecules exhibit a different pattern of subcellular localization. Incubation with sialidase revealed the existence of hidden L-selectin epitopes at the cell surface, while no additional Mac-1 epitopes were exposed. Platelet-activating factor stimulation decreased surface and total expression of L-selectin to the same extent in both populations, but solely affected Mac-1 surface expression on eosinophils. Moreover, cytoskeletal actin filaments and microtubules were found to be involved in the regulation of Mac-1 surface expression on bovine neutrophils and eosinophils. In marked contrast, expression of L-selectin was minimally affected by cytoskeleton perturbing agents. The present study indicates that L-selectin and Mac-1 adhesion molecules reside in distinctly located reservoirs in bovine granulocytes and can be selectively mobilized upon in vitro stimulation.     

Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood

An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals. At present, only a few tests are available for the diagnosis of these latent M. suis infections in pigs. The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M. suis-infected pigs. A 1.8 kb EcoRI DNA fragment of the M. suis genome was isolated from the blood of pigs experimentally infected with M. suis. Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment. PCR products of 782 bp in size were amplified only from M. suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas. PCR results were confirmed by dot blot hybridisation. The applicability of the PCR assay to diagnose M. suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs. The M. suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals.     

Genetic analysis of markers of the performance test for herding dogs. 1. Performance traits

The objective of the present study was to analyse data based on records of sheep dog trials in order to evaluate the importance of genetic and environmental sources of variation and to draw conclusions for breeding purposes. The data analysed consisted of 2745 test results recorded at 48 sheep dog trials carried out in Germany from 1994 to 1998, on which a total of 337 sheep dogs had attended. Tested dogs descended from 147 sires and 201 dams, with a mean inbreeding coefficient of about 2.8%. Variance components of working traits were estimated applying Restricted Maximum Likelihood methods. Additive genetic effects, permanent environmental effects of the animal and the effect of the handler were treated as random factors using multivariate linear models. Additionally, the model included the fixed effects of the age of the dogs at the sheep dog trial, sex, the level of difficulty of the exercises as well as the event itself, the starting number of the dog, the number of the dogs' tests at the particular event and the number of dogs presented on sheep dog trials by their handlers. The inbreeding coefficient was regarded as a linear covariate. Sex of the dog and starting number did not explain a significant proportion of variance for traits analysed, whereas the event of the sheep dog trial and the starting class usually were of significant importance. The analyses were performed using all sheep dog trial classes and for each of the three classes separately. Repeatabilities of herding traits within the same event of sheep dog trial estimated from animal effect mostly varied within a range from w = 0.1 to w = 0.5 whereas repeatabilities estimated over all events of sheep dog trials mostly gave estimates from w = 0.1 to w = 0.3. The estimated heritabilities for the herding performance traits ranged from h2 < or = 0.001 to h2 = 0.13 with standard errors in the range between 0.001 and 0.08. The selection possibilities appear to be rather limited given the heritability estimates and the low number of progeny of parents.     

Genetic analysis of markers of the working test for herding dogs 2. Undesired behavior traits

The objective of the present study was to analyse the occurrence of undesirable behaviour traits registered on sheep dog trials in order to evaluate the importance of genetic and environmental sources of variation and to draw conclusions for breeding purposes. The data analysed consisted of 2745 test results recorded at 48 sheep dog trials carried out in Germany from 1994 to 1998, which were attended by 337 sheep dogs. Variance components of undesirable behaviour traits were estimated applying Restricted Maximum Likelihood methods. Additive genetic effects, permanent environmental effects of the animal and the effect of the handler were treated as random factors. Additionally, the linear multivariate animal model included the fixed effects of the age of the dogs at the sheep dog trial, sex, the level of difficulty of the exercises as well as the event itself, the starting number of the dog, the number of the dogs' tests at the particular event and the number of dogs presented on sheep dog trials by their handlers. The inbreeding coefficient was regarded as a linear covariate. The analyses were performed using all sheep dog trial classes and for each of the three classes separately. Age of the dog and starting number did not explain a significant proportion of variance for traits analysed, whereas the event of the sheep dog trial and partly the number of dogs per handler were of significant importance. The estimated heritabilities for the undesirable behaviour traits ranged from h2 < or = 0.001 to h2 = 0.07 with standard errors in the range between 0.001 and 0.06. The possibilities to select against undesired behaviour traits appear to be rather limited given the heritability estimates and the low number of progeny.     

Apoptosis of bovine neutrophils during mastitis experimentally induced with Escherichia coli or endotoxin

OBJECTIVE: To determine whether apoptosis of neutrophils was accelerated during mastits experimentally induced by use of Escherichia coli or E coli endotoxin and whether differences were apparent in the response to E coli or endotoxin. ANIMALS: 11 healthy lactating Holstein cows. PROCEDURE: Blood samples were collected from cows at various intervals after intramammary inoculation with E coli or endotoxin. Percentage of apoptotic neutrophils detected after in vitro incubation for 3 hours was determined. Fluorescein isothiocyanate-labeled annexin-V in combination with propidium iodide was used to distinguish apoptosis and necrosis of neutrophils. Total and differential circulating leukocyte counts and rectal temperature were determined at the time of collection of blood samples. Milk yield and milk somatic cell counts were determined at the time of milking. RESULTS: Inoculation of endotoxin did not accelerate in vitro induction of neutrophil apoptosis. However, inoculation of E coli increased the percentage of apoptotic neutrophils. At 18 hours after inoculation, 20% of the neutrophils were apoptotic, compared with 5% before inoculation. Milk somatic cell count and rectal temperature increased, milk production and total leukocyte count decreased, and percentage of immature neutrophils increased after inoculation with E coli or endotoxin. However, kinetics of the responses were more rapid, more severe, and of shorter duration during endotoxin-induced mastitis. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro induction of apoptosis of neutrophils was accelerated only during E coli-induced mastitis and not during endotoxin-induced mastitis. Endotoxin inoculation as a model for studying coliform mastitis in dairy cows should be viewed with caution.     

Recombinant bovine soluble CD14 sensitizes the mammary gland to lipopolysaccharide

Standard therapies including administration of potent antibiotics, aggressive fluid resuscitation and metabolic support have not been successful in relieving symptoms and reducing mortality associated with acute coliform mastitis. It is important to understand the pathophysiological response of the mammary gland to coliform infections when designing preventive or therapeutic regimens for controlling coliform mastitis. Our laboratory has previously shown that macrophages and polymorphonuclear neutrophils in milk express CD14 on their cell surface. In this study, we found that soluble CD14 (sCD14) is present in milk whey as a 46kDa protein reacted with anti-ovine CD14 antibody. Additional functional studies found that: (1) under serum-free condition, complexes of LPS-recombinant bovine soluble CD14 (rbosCD14) induced activation of mammary ductal epithelial cells (as measured by changes in interleukin-8 (IL-8) mRNA level by competitive RT-PCR) at low concentrations of LPS after 6 or 24h incubation (1-1000ng/ml), whereas LPS alone did not induce activation of mammary ductal epithelial cells at the same concentrations, and (2) intramammary injection of low concentrations of LPS did not increase concentration of leukocytes in milk. In contrast, LPS-rbosCD14 complex containing the same concentration of LPS increased the concentration of leukocytes in the injected mammary gland at 12 and 24h post-injection. These results indicate that rbosCD14 sensitizes mammary epithelial cells to low concentrations of LPS in vitro and in vivo. Endogenous sCD14 in milk may be important in initiating host responses to Gram-negative bacterial infections.