Typing of Legionella isolates during an epidemiological investigation can be helpful but also misleading: an example from Greece.
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Denmark↗
Biomedical subjects
S S Nielsen
Publications and source records attributed to S S Nielsen.
Cyclooxygenase type 2 is increased in obstructed rat and human ureter and contributes to pelvic pressure increase after obstruction.
Prostanoids exert physiological effects on ureteral contractility that may lead to pressure changes and pain during obstruction. In the present study, we examined whether (1) obstruction changes the expression of the two cyclooxygenase (COX) isoforms, COX-1 and COX-2 in human and rat ureters and (2) administration of a selective COX-2 inhibitor influences the pelvic pressure change after experimental ureteral obstruction. Rats were subjected to bilateral ureter obstruction. Ureters were removed and dissected into a proximal dilated and distal non-dilated segment. RNA and protein were extracted and analyzed for cyclooxygenase expression by quantitative polymerase chain reaction and Western blotting. Human ureter samples were obtained from patients undergoing radical nephrectomy. Rat and human ureteral samples were processed for immunohistochemistry. COX-1, but not COX-2 mRNA, was readily detected in the normal rat ureter. COX-2 mRNA and protein expression was increased in the proximal dilated ureter compared to distal non-dilated ureter. This increased COX-2 expression was associated with increased urinary prostaglandin E2 (PGE2) excretion after release of obstruction. Immunohistochemistry showed increased COX-2 labeling in surface epithelium and smooth muscle layers in both rat and human obstructed ureters compared to control ureters. Furthermore, contractile PGE2-EP1 and thromboxane TP receptors were expressed in ureteral smooth muscle. Systemic treatment with the COX-2 selective inhibitor parecoxib (5 mg/kg/day) attenuated the pelvic pressure increase during obstruction. In summary, COX-2 expression is significantly increased in the ureteral wall in response to obstruction in the rat and human ureter and COX-2 activity contributes to increased pelvic pressure after obstruction.
Animals↗
Host-specific bacterial lineages in the taxon 2 and 3 complex of Pasteurellaceae.
The aim of the investigation was to determine the genetic relationship of a phenotypically diverse strain collection of epidemiologically unrelated strains from the taxon 2 and 3 complex of Bisgaard isolated from different hosts. A total of 325 isolates belonging to the taxon 2 and 3 complex of Bisgaard was characterized phenotypically in 82 characters. The genetic relationship among a subset of 60 isolates was investigated by amplified fragment length polymorphism (AFLP). The isolates were selected aiming at including the broadest diversity with regards to phenotype and host spectrum. The results suggested a statistically clear association between AFLP clusters and the host species families Columbidae (pigeon, dove), Anatidae (duck, goose) and Psittacidae (parrot, parakeet, budgerigar), respectively. This association was further supported by results from previous whole cell protein profiling and DNA:DNA hybridization studies. In conclusion, it appears that distinct genetic lineages within the taxon 2 and 3 complex of Bisgaard have evolved specificity for host bird species of different families. The observed host specificity of taxon 2 and 3 organisms may be used in future diagnostics and studies elucidating aspects of pathogenicity and epidemiology associated with the different lineages and their respective hosts.
Adaptation, Biological↗
Age-specific characteristics of ELISA and fecal culture for purpose-specific testing for paratuberculosis.
Paratuberculosis is a chronic infection, and animals are not equally affected by it. Therefore, diagnostic tests that are able to detect different stages of the infection are needed for objective decision making. A longitudinal study was carried out to describe the ability of 2 tests to predict 2 conditions in dairy cattle: "infection" and "infectious," exemplifying 2 different purposes of testing. "Infection" is the term of choice for certification and eradication purposes, and "infectious" is more relevant for control purposes. In the study period of 3 yr, repeated sampling of milk (n = 23,219) and feces (n = 8,832) was performed. A total of 1,985 Danish dairy cows provided material for the study. Milk samples were analyzed for antibodies using an ELISA, and fecal samples were analyzed for mycobacteria by culture. A reference test to correctly classify cattle antemortem does not exist; thus, "infection" and "infectious" were defined by repeated testing using one test as the condition to be detected by the other test. Fecal culture responses were evaluated against antibody status, and ELISA responses were evaluated against detected bacterial shedding. The results of this study indicate that the ability of both tests to detect "infection" increases almost linearly from 2 to 5 yr of age, whereas the ability of both tests to detect "infectious" is not affected by age. Purpose-specific tests are required to appropriately interpret and use test results for management of paratuberculosis, and relevant covariates, such as age, should be included when possible.
Aging↗
Activity and nature of plasminogen activators associated with the casein micelle.
In fresh milk, plasminogen, the zymogen form of plasmin (PL), is the predominant form. Therefore, plasminogen activators (PA) can contribute significantly to PL activity in milk. Both tissue-type PA (tPA) and urokinase-type PA (uPA) exist in milk; however, contradictory findings have been reported for which type of PA is most closely associated with the casein micelles. Little is known about the factors that might lead to variations in the individual activities of the PA. The objective of this work was therefore to investigate possible factors that might affect the association of tPA and uPA with the casein micelle and their activities thereafter. Plasminogen activators were isolated from milk samples with different somatic cell counts following 2 different isolation protocols. Determination of uPA, tPA, and PL activities was carried out quantitatively following chromogenic assays using 2 different substrates, and qualitatively using specialized sodium dodecyl sulfate-PAGE. Different isolation methods and conditions led to differences in uPA, tPA, and PL activities. Urokinase-type PA activity was significantly higher in PA fractions isolated from milk with high somatic cell counts than from milk with low somatic cell counts. Activity results indicated that in pasteurized milk uPA could dissociate from the somatic cells and bind to casein. Moreover, a high level of PL in isolated PA fractions contributed to significantly enhanced PA activities. Overall, results confirmed the association of both uPA and tPA with the casein micelle; however, their amounts, activities, and molecular weights varied based on the nature of the milk and methods of separation, with uPA being the PA with greater potential to affect plasminogen activation in milk.
Animals↗
Age at occurrence of Mycobacterium avium subspecies paratuberculosis in naturally infected dairy cows.
Paratuberculosis is a chronic infection of ruminants and other species caused by Mycobacterium avium ssp. paratuberculosis (Map). Establishing test strategies for paratuberculosis will require insight into the temporal aspects of certainty with a given test. In this study, the age at which cows tested positive by ELISA and fecal culture (FC) was investigated by use of time-to-event analyses. The effects of herd, parity, and shedding group were evaluated at the age of test-positive ELISA and FC, respectively. Finally, the test frequency was investigated for the probability of cows being tested ELISA-positive. Milk and fecal samples were collected repeatedly over a 3-yr period from 1,776 Danish dairy cows from 8 herds. The milk samples were tested for the presence of antibodies by using an ELISA, and an FC test was used for detection of Map. Repeated ELISA testing detected 98 and 95% of cows classified as high and low shedders, respectively, suggesting that most infected cows develop antibodies. Among the high shedders, 50% were positive before 4.3 yr of age (quartiles 1 to 3: 3.4 to 5.7 yr of age). Repeated FC detected only 72% of the cows that were ELISA-positive, and 50% of the ELISA-positive cows were detected by FC at 7.6 yr of age. The age with the highest probability of testing positive was determined as the interval with the steepest slope in the survival probability plots. The highest probability of testing positive by ELISA was from 2.5 to 4.5 yr of age. The highest probability of testing positive by FC was from 2.5 to 5.5 yr of age. For both ELISA and FC, testing positive was highest in the first 300 d in milk. For cows younger than 4 yr of age, monthly testing with ELISA, compared with testing every 2 yr, could increase the probability of detecting cows with antibodies by 19%. In older cows, there were no apparent differences in the probability of testing positive by monthly sampling compared with sampling every second year. Therefore, for older animals the effect of more frequent sampling would be for early detection rather than to obtain additional information. Cows shedding high numbers of Map will produce antibodies, although not necessarily concomitantly with the shedding. These antibodies can be detected by ELISA with a test strategy that is different for younger and older cows. We suggest testing younger cows more frequently than older cows and that testing should be done prior to 350 d in milk.
Age Distribution↗
Estimating receiver operating characteristic curves with covariates when there is no perfect reference test for diagnosis of Johne's disease.
Paratuberculosis (Johne's disease) is a significant animal health problem. Evaluation of diagnostic tests for Johne's disease has been difficult due to lack of a gold standard test. In recent years, there has been interest in receiver operating characteristic (ROC) curve estimation without any gold standard test. Typically, either Bayesian or maximum likelihood methods are proposed. Although these methods overcome the lack of a gold standard test in ROC curve estimation, little work has been done to incorporate covariates in the analysis. In this paper, we propose a method for estimation of ROC curves based on statistical models to adjust for covariate effects when the true disease states of test animals are unknown. The covariates may be correlated with the disease process or with the diagnostic testing procedure, or both. We propose a 2-part Bayesian model: first, a logistic regression model for disease prevalence is used to fit the covariates; second, a linear model is used to fit the covariates to the distribution of test scores. We used Markov chain Monte Carlo methods to compute the posterior estimates of the sensitivities and specificities that provide the groundwork for inference concerning the diagnostic procedure's accuracy. We applied the methodology to milk ELISA scores from several dairy-cow herds for the diagnostic testing of paratuberculosis. We found that both milk yield and its interaction with age had significant effects on the disease process whereas only milk yield was significant on the testing procedure.
Animals↗
Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests.
Three recombinant soybean cysteine proteinase inhibitors (rSCPIs), L1, R1 and N2, were assessed for their potential to inhibit the growth and development of three major agricultural crop pests known to utilize digestive cysteine proteinases: Western corn rootworm (Diabrotica virgifera virgifera, WCR), Colorado potato beetle (Leptinotarsa decemlineata, CPB) and cowpea weevil (Callosobruchus maculatus, CW). In vitro experiments showed that cysteine proteinase activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR, CPB, and CW (50% inhibition at 5 x 10(-8), 5 x 10(-8), and 3 x 10(-7) M, respectively). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 x 10(-6) M of the crude gut extracts of WCR. Results of in vivo experiments conducted to assess the effect of the three rSCPIs on the vital growth parameters of WCR, CPB and CW were consistent with results of the in vitro experiments.
Animals↗
Histopathology of oligofructose-induced acute laminitis in heifers.
Histopathology of the dermo-epidermal junction in the lamellar region of front claws was examined in 6 dairy heifers given an alimentary oligofructose overload and compared with sections from a control group of 6 heifers. Four of the 6 heifers administered oligofructose developed clinical signs of acute laminitis before they were euthanized. Postmortem samples from front claws were processed for histology. Eleven histopathologic characteristics were selected from the existing literature and used in a blinded evaluation of sections. In total, 104 front claw samples, including 8 samples from 2 cows having spontaneously occurring acute laminitis, were evaluated histologically using hematoxylin and eosin as well as periodic acid-Schiff staining. The major morphological features associated with oligofructose-induced acute clinical laminitis were stretching of lamellae, dermal edema, hemorrhage, changes in basal cell morphology, presence of white blood cells in dermis, and signs of basement membrane detachment. Changes at the lamellar junction of claw tissue affected by oligofructose-induced clinical laminitis resembled tissue from the 2 cows suffering from spontaneous acute clinical laminitis, and generally were consistent with existing descriptions of laminitis histopathology. Important exceptions to existing descriptions in the literature were stretching of lamellae and basement membrane changes. Not previously described, we considered these early signs of acute laminitis. In conclusion, this study documents that oligofructose-induced clinical laminitis is associated with histopathological changes at the lamellar interface. A weakened dermo-epidermal junction is a possible intermediate stage in the pathophysiology of bovine sole ulceration at the typical site.
Animals↗
Effects of Pseudomonas fluorescens M3/6 bacterial protease on plasmin system and plasminogen activation.
Heat-stable proteases produced by the psychrotroph Pseudomonas fluorescens M3/6 have been shown to affect the plasmin system in milk, which in turn will affect the quality of processed milk. The M3/6 proteases cause dissociation of plasmin from casein in minimally processed milk. The objective of this work was to study the effect of M3/6 protease on the plasmin system, as well as its role in plasminogen activation, under commonly applied cheese-making conditions. Isolated M3/6 protease was added to raw milk, which then was pasteurized, and subjected to pH adjustments and CaCl2 addition. Casein and whey fractions were separated by chymosin treatment then analyzed for plasmin activity. Individual and interaction effects of M3/6 protease addition, pH treatment, and CaCl2 addition on plasmin activity were studied. Enzyme activity assays were carried out to study individually the effect of M3/6 protease on plasmin system components. Kinetic parameters were calculated to characterize the effect of M3/6 protease on plasminogen activation. Plasmin activity increased in the curd fractions of the protease-treated milk that was subjected to conditions most resembling cheese-making conditions, indicating that M3/6 protease triggered plasminogen activation rather than dissociation of plasmin from casein micelles. Results from the studies on plasminogen activation confirmed that the observed activation of plasminogen in protease-treated samples subjected to cheese making conditions was attributed to the stimulatory effect M3/6 protease had on plasminogen activators (PA). The M3/6 protease stimulated human and bovine PA by increasing their activity 4.5- and 2.5-fold, respectively. Similarly, the catalytic efficiencies of human urokinase-type PA and bovine PA were increased in the presence of M3/6 protease by 12- and 4-fold, respectively. Our research presented a basic step toward fully understanding the effect of bacterial proteases under different processing conditions, where the gathered information can aid in better control of processing conditions based on the desired outcome.
Animals↗
Continuous-data diagnostic tests for paratuberculosis as a multistage disease.
We devised a general method for interpretation of multistage diseases using continuous-data diagnostic tests. As an example, we used paratuberculosis as a multistage infection with 2 stages of infection as well as a noninfected state. Using data from a Danish research project, a fecal culture testing scheme was linked to an indirect ELISA and adjusted for covariates (parity, age at first calving, and days in milk). We used the log-transformed optical densities in a Bayesian network to obtain the probabilities for each of the 3 infection stages for a given optical density (adjusted for covariates). The strength of this approach was that the uncertainty associated with a test was imposed directly on the individual test result rather than aggregated into the population-based measures of test properties (i.e., sensitivity and specificity).
Aging↗
Relationship between antibodies against Mycobacterium avium subsp. paratuberculosis in milk and shape of lactation curves.
To analyze how infection with Mycobacterium avium subsp. paratuberculosis (MAP) affects the shape of lactation curves, a three-level hierarchical test-day model was set up with fat-corrected test-day milk yield (FCTM) as response. Milk samples from 6955 cows in 108 Danish dairy herds were tested with ELISA to detect antibodies against MAP. Optical densities (ODs) recorded on a continuous scale were standardized according to parity and stage of lactation. In addition to standardized ODs (stOD), seven fixed covariates, quadratic terms and first-order interactions were included in the model. Cow and cow nested in herd were included as random effects. Cows of first, second and higher parities were analyzed separately. The lactation curves after peak yield were significantly less persistent in young infected cows, where an increase of one stOD unit was associated with a depression of the milk yield per day through day 305 of 3.7 kg FCTM in first parity and 2.7 kg FCTM in second parity. In second-parity cows, the lactation curve also was both depressed through the entire lactation and more steep after 60 days in milk (DIM). In third and older parities, a significant effect of the quadratic term of stOD indicated exponentially increased losses with increased ODs.
Animals↗
Comparison of contamination and growth of Mycobacterium avium subsp. paratuberculosis on two different media.
AIMS: The purpose of the study was to compare the growth of Mycobacterium avium subsp. paratuberculosis (Map) and the degree of contamination on Herrold's egg yolk medium (HEYM) and modified Löwenstein-Jensen medium (LJ). METHODS AND RESULTS: Culture of 2513 faecal samples from dairy cows was performed on each of the two media. The media were read after 5, 8 and 12 weeks of incubation. Overall, the proportion of contaminated samples was significantly higher on LJ (14.2%) than on HEYM (13.2%) after 12 weeks but the degree of contamination was slightly less on LJ. After 8 weeks of incubation, only 1.0% of the samples were Map positive in LJ with 4.9% on HEYM. After 12 weeks of incubation, 3.3% of the samples were Map positive in LJ whereas 6.9% were positive in HEYM. All suspect and culture positive samples were confirmed by IS900 PCR. CONCLUSIONS: HEYM supported growth of Map significantly better and faster than LJ, however it could not be determined conclusively which of the two media that provided the highest degree of decontamination when the incubation time was also included. SIGNIFICANCE AND IMPACT OF THE STUDY: HEYM should be the primary medium rather than LJ for detection of Map in cattle.
Animals↗
Genetic variation and heritability of the antibody response to Mycobacterium avium subspecies paratuberculosis in Danish Holstein cows.
The purpose of this study was to estimate the genetic variation and the heritability of the ability to establish an immune response by producing antibodies to Mycobacterium avium subsp. paratuberculosis. Antibody levels were determined using an ELISA and measuring optical density (OD) values from milk samples of 11,535 cows from 99 herds. The pedigree of the 11,535 cows and information about days in milk, parity, milk yield, and others were obtained from the Danish Cattle database. The statistical analyses were made using a bivariate mixed animal model. The bivariate model with daily milk yield and OD as dependent variables showed a significant heritability of the ability to produce Mycobacterium avium subsp. paratuberculosis antibodies of 0.102 (genetic variance=0.054) and a nonsignificant genetic correlation of -0.037 between daily milk yield and OD. When a sire model was used, the estimated heritability was 0.091. To evaluate whether intrauterine infection could be reflected in the OD, variation among 2715 dam-daughter or maternal sister groups was also estimated. Variation in this data was nonsignificant, possibly because only very few clinical cases or end-stage cows, i.e., heavy bacterial shedders may have been represented among the cows sampled. It does not appear that intrauterine transmission is of major importance in the transmission of paratuberculosis.
Analysis of Variance↗
Effects of native and denatured whey proteins on plasminogen activator activity.
The plasmin system native to bovine milk consists of the caseinolytic serine proteinase plasmin; its inactive zymogen, plasminogen; plasminogen activators; and inhibitors. Evidence in the literature indicates that whey proteins may inhibit plasmin activity, but there is very little mention of their effect on plasminogen activators. The objective of this research was to determine the effect of both unheated and heat-denatured beta-lactoglobulin (beta-LG), alpha-lactalbumin (alpha-LA), and BSA on plasminogen activators. Plasminogen activator activity was significantly stimulated by non-heat treated and denatured alpha-LA as well as by denatured beta-LG. The stimulation effect by these whey proteins was kinetically characterized, which showed that all 3 significantly increased the rate of plasminogen activation. The stimulation effect was shown to be independent of any effect of the whey proteins on plasmin activity by testing 2 different substrates, d-Val-Leu-Lys p-nitroanilide (S-2251) and Spectrozyme PL (Spec PL), in a plasmin assay. Results using S-2251 confirmed the inhibitory effect of whey proteins on plasmin observed by several researchers. However, use of SpecPL did not suggest inhibition. Ligand binding studies showed this discrepancy to be due to significant interaction between S-2251 and the whey proteins. Overall, this study indicates that whey protein incorporation into cheese may not hinder plasmin activity and may stimulate plasminogen activation. Furthermore, the results indicate the need for careful consideration of the type of synthetic substrate chosen for model work involving whey proteins and the plasmin system.
Fibrinolysin↗
Variance components of an enzyme-linked immunosorbent assay for detection of IgG antibodies in milk samples to Mycobacterium avium subspecies paratuberculosis in dairy cattle.
Milk samples from 120 cows were tested up to 10 times in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycobacterium avium ssp. paratuberculosis. The purpose of the study was to estimate variance components of the assay attributable to laboratory factors using mixed model theory. Because of significant interaction between the between-run, between-day and between-plate variables, the ELISA-plate variable was nested in run-number and run-number was nested in day-number. The nested variable accounted for 68% of the laboratory variability (P < 0.001), whereas the intraplate variability accounted for only 0.04% of the laboratory variability (P > 0.9). Therefore, it was concluded that the intraplate variability could be ignored whereas the variability from the combined run-day-plate variable should be considered in any analyses based on the ELISA.
Animals↗
Patients with ovarian cancer have elevated (51)Cr-EDTA plasma clearance early post-operatively.
Plasma clearance of (51)Cr-EDTA (Clp(EDTA)) is widely used to determine glomerular filtration rate prior to carboplatin based chemotherapy. We have observed that many patients with ovarian cancer have elevated Clp in the early post-operative phase compared to later phases. The purpose of this study was to examine whether this observation reflects a systematic difference. We retrospectively analysed data from 53 patients who had undergone surgery for ovarian cancer. Twenty-six patients had Clp(EDTA) measured early after the operation (mean, 8 days (range, 3-16 days)) (early group), and 27 patients had Clp(EDTA) measured late post-operatively (mean, 32 days (range, 19-48 days)) (late group). Clp(EDTA) values was measured before the first, third and fifth course of chemotherapy. Additionally, age, height, weight, cancer stage, ascites and tumour histology were noted. Mean Clp(EDTA) in the early group was significantly higher than in the late group (104+/-4.4 vs 89+/-3.5 ml.min(-1) per 1.73 m(2); P =0.005). Clp(EDTA) declined significantly in the early group from the first measurement after the operation until measurement before the third course of chemotherapy but remained constant in the late group. Clp(EDTA) was not correlated to ascites, cancer stage or tumour histology. It is concluded that patients with ovarian cancer have significantly higher Clp(EDTA) in the early post-operative phase than similar patients with Clp(EDTA) measured late post-operatively.
Antineoplastic Agents↗
Paratuberculosis in dairy cattle: variation of the antibody response in offspring attributable to the dam.
The objective of this study was to examine transmission of paratuberculosis in dairy cattle attributable to the dam. Milk samples were collected from 8131 cows in 110 Danish dairy herds. The level of antibodies to Mycobacterium avium subspecies paratuberculosis was determined by use of an ELISA. Information on dam and sire was obtained from the Danish Cattle database. The following two data sets were analyzed: Data set A contained all cows < or = 400 days in milk (n = 7410); data set B contained 1056 dam-daughter pairs present simultaneously in herds at the day of sampling. Cows > 400 days in milk were excluded. Linear mixed models were used to obtain variance components for the effect of sire in data set A and the effect of sire and dam-daughter pairs in data set B. Models for both data sets A and B included information previously shown to confound antibody level and information of the relative prevalence of paratuberculosis in the herd. In data set A, the effect explained by sire was 1.9%, whereas it was 6.3% in data set B. The effect from dam-daughter pairs was 7.7%. Those effects were all significant. It was concluded that the parental contribution was significant, and both heritability of susceptibility and vertical transmission should be considered in any control programs on paratuberculosis in dairy cattle.
Animals↗