Plasma endotoxin activity in Eastern grey kangaroos (Macropus giganteus) with lumpy jaw disease.

Progressive pyogranulomatous osteomyelitis involving the mandible or maxilla of captive macropods, referred to as "Lumpy jaw disease (LJD)", is one of the most significant causes of illness and death in captive macropods. The aim of the present study was to evaluate the relationship between the severity of LJD and plasma endotoxin activity in kangaroos. Plasma samples obtained from moderate (n=24) and severe LJD (n=12), and healthy kangaroos (n=46), were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. Plasma endotoxin activity was measured using the Limulus amebocyte lysate (LAL)-kinetic turbidimetric (KT) assay. Plasma endotoxin activity was higher in kangaroos with severe LJD (0.199 ± 0.157 EU/ml) than in those with moderate LJD (0.051 ± 0.012 EU/ml, P<0.001) and healthy controls (0.057 ± 0.028 EU/ml, P<0.001). Our results suggest that the severity of LJD in captive macropods may be related to the plasma endotoxin activity.

Evaluation of a portable test system for assessing endotoxin activity in raw milk.

The aim of the present study was to compare endotoxin activities detected in raw milk samples obtained from cattle by a commercially available portable test system (PTS) and traditional microplate limulus amebocyte lysate (LAL)-based assay, which determined activities using a kinetic turbidimetric (KT) assay. Raw milk samples were obtained from 53 and 12 dairy cattle without and with clinical mastitis, respectively. Comparison between the KT and PTS was performed by the Friedman test. The Pearson product moment correlation coefficients were calculated to evaluate associations between any two continuous variables. Linear regression model analysis was also performed to obtain the equation describing the relationship between PTS and KT assay. The endotoxin activities detected in 200- or 400-fold diluted milk samples were similar between PTS and KT assay, whereas a significant difference was observed in 100-fold diluted milk (P<0.001). The results obtained from 200- (r(2)=0.778, P<0.001) and 400-fold diluted milk samples (r(2)=0.945, P<0.001) using PTS correlated with those using KT assay. The median milk endotoxin activities in Gram-positive and Gram-negative clinical mastitis cows were 0.655 and 11,523.5 EU/ml, respectively. The results of the present study suggest that PTS as a simple and easy test to assess endotoxin activity in raw milk is efficient, simple and reproducible.

Determination of the inflammatory potential of bioaerosols from a duck-fattening unit by using a limulus amebocyte lysate assay and human whole blood cytokine response.

Inhalation of bioaerosols from animal houses can induce acute inflammatory reactions in the respiratory tract. Determination of the concentration of airborne endotoxins is widely used to characterize this risk. In this study, the activity of bioaerosol samples from a duck-fattening unit to induce interleukin-1beta (IL-1beta) in human blood and to react with Limulus Amebocyte Lysate (LAL) was investigated. The activity detected in the whole blood assay correlated well with the endotoxic activity found in the LAL assay (Spearmen's rho = 0.902). However in all samples, the inflammation-inducing potential was overestimated by the LAL assay. It is assumed that this overestimation could be, in part, a result of an overestimation of the inflammatory potential of endotoxins originating from Pseudomonadaceae by the LAL assay. Pseudomonadaceae were regularly isolated from the air of the duck-fattening unit. The results presented here indicate that the whole blood assay can be used besides the LAL assay as an additional method to characterize the inflammation-inducing potential of bioaerosols.

Plasma endotoxin concentration in horses: a methods study.

BACKGROUND: Accurate determination of plasma endotoxin concentration is critical for ex vivo and in vitro cellular and molecular studies of endotoxemia in horses. However, reports are conflicting with respect to anticoagulant, handling, and sample preparation. OBJECTIVE: The purpose of this study was to determine the effect of blood sample fraction and handling time on measurement of endotoxin concentration in horses. METHODS: Whole blood, anticoagulated with 3.8% (0.12 M) sodium citrate (9:1), was collected from 5 healthy horses. Whole blood (WB), platelet-rich plasma (PRP), and platelet-poor plasma (PPP) were spiked with endotoxin (2 EU/mL). Endotoxin-spiked WB samples were centrifuged immediately to generate PRP for measurement. Endotoxin concentration was subsequently measured by Limulus amebocyte assay at 0, 15, 30, 45, and 60 minutes. Assays were performed in triplicate and results were analyzed using Student's t-test, with significance set at P <.05. RESULTS: Mean endotoxin concentrations in 2 EU/mL-spiked WB were significantly different from those in PPP at all time points tested. Recovery of endotoxin in PRP generated from WB was significantly diminished after just 15 minutes. CONCLUSION: PRP generated from WB is significantly more reliable than PPP in determining endotoxin concentration ex vivo. Measurement of endotoxin in PRP generated from WB was significantly diminished after 15 min, identifying a time frame within which to process blood samples for endotoxin analysis.

Bacterial colonization and endotoxin activity during experimental acute fowl typhoid in chickens.

Bacterial colonization and endotoxin production were investigated before and after experimental Salmonella gallinarum infection in 8-week-old female broiler chickens. These parameters were assayed by means of colony forming units test (CFU) and the Limulus Amebocyte Lysate test (LAL), respectively. Birds were infected per os with 1,5 x 10(9) CFU/ml of wild strain of S. gallinarum isolated from a dead hen. Approximately 1,5 x 10(2); 1,3 x 10(2) and 1,2 x 10(2) CFU of S. gallinarum were recorded from 1 g of liver, 1 g of spleen and 1 ml of blood from the chickens on day 1 post infection. By day 4 corresponding data were 3,7 x 10(4); 4,8 x 10(3) and 1,1 x 10(3) respectively and on day 7 10(5) CFU were present in all three specimen types. The liver and spleen of dead birds were contaminated with more than 10(7) CFU per g. The endotoxin from S. gallinarum was found to have an activity of 1,5; 12,0 and 15,0 endotoxin units (EU)/ml on day 1, 4 and 7 after infection, respectively. No endotoxin activity was established in the blood of the control group (before infection) by the LAL test. This is the first time the connection between the amount of live S. gallinarum in the blood, liver and the circulating level of endotoxin in the blood during the infectious stage of experimental acute fowl typhoid, has been demonstrated.

Biological activities of lipopolysaccharides extracted from porcine vaccine strains.

Lipopolysaccharides (LPSs) were purified from Actinobacillus pleuropneumoniae serotype 2, Bordetella bronchiseptica and Haemophilus parasuis serotype 5, which were used for vaccine production in Japan, by the phenol-water procedure. In SDS-PAGE analysis, A. pleuropneumoniae LPS, as well as Escherichia coli LPS, demonstrated a typical ladder profile of a smooth-type LPS. On the other hand, B. bronchiseptica and H. parasuis LPSs lacked the ladder profiles. It was found that the biological activity of these LPSs was comparable to those of E. coli LPS in terms of activation of the clotting enzyme of Limulus amoebocyte lysate, mitogenic activity of mouse spleen cells, stimulation of TNF-alpha and nitric oxide production, but IL-6 production could hardly be observed in any LPS.

Endotoxin-induced platelet aggregation in heparinised equine whole blood in vitro.

Endotoxaemia is a leading cause of death among horses. Thrombocytopenia is a common finding in clinical and experimentally-induced cases of endotoxaemia and can lead to coagulopathies, including disseminated intravascular coagulopathy which is usually fatal. In this study it was shown that endotoxin (3 ng ml-1 to 25 micrograms ml-1) can aggregate equine platelets in heparinised whole blood in vitro. The endotoxin-induced aggregation (EIA) was shown to be dependent on the presence of leucocytes in the blood and did not occur when detoxified endotoxin was used, suggesting that lipid A was necessary for the response. Aspirin (1 mmol litre-1) had no effect on EIA whereas apyrase (40 micrograms ml-1) completely abolished it and CV3988 (3 to 30 mumol litre-1) (a competitive antagonist of platelet-activating factor) inhibited the response in a concentration-dependent manner. It is concluded that endotoxin activates equine platelets at low concentrations through an indirect mechanism that involves calcium, leucocytes, adenine nucleotides and platelet-activating factor.

Effect of feeding regimen on concentration of free endotoxin in ruminal fluid of cattle.

The influence of concentrate diets on endotoxin concentration in sterile filtrate of ruminal fluid was assessed in ruminally fistulated Jersey cows. Three cows underwent a change in diet from hay to a diet containing 3.0 kg and 14 d later 6.0 kg of a 12% CP concentrate. The cows had free access to water and a mineral-stone. A modified Limulus Amoebocyte Lysate technique was used for the endotoxin analyses. The endotoxin concentration in the ruminal fluid of cows fed on hay were 148 +/- 84 and 118 +/- 50 endotoxin units (EU)/mL (mean +/- SD) on two separate days and increased from the 2nd d of supplementation with 3.0 kg of concentrate to 408 +/- 198 EU/mL on d 5. When the cows were fed 6.0 kg of concentrate, the endotoxin concentrations increased to 1,599 +/- 944 EU/mL. To assess the influence on the ruminal endotoxin concentration of an adaptation of the ruminal environment to concentrate before hyperalimentation, four ruminally fistulated Jersey cows previously fed either hay or a high-concentrate diet for 1 mo were hyperalimented with 60 to 70 g of barley per kilogram BW. A relative increase in ruminal endotoxin concentration was determined only in the two cows previously fed concentrates. The results show that high-concentrate diets do not consistently relate to increases of ruminal endotoxin concentrations.

Evaluation of a cow-side test for detection of gram-negative bacteria in milk from cows with mastitis.

A modified Limulus amebocyte lysate (LAL) cow-side test was evaluated under field conditions. The principle of the test is to visualize reactions between test components and endotoxin from the cell wall of Gram-negative bacteria. The practical purpose is to detect such bacteria in mastitic milk. Secretions from 789 udder quarters with clinical mastitis were examined by the LAL-test. Parallel quarter milk samples were sent to a mastitis laboratory of microbiological examination. Eleven veterinary surgeons in three veterinary districts in Norway performed the field investigations. Results of the LAL-test and culture agreed in 93% of all milk samples tested, agreement measured by kappa being 0.63. The sensitivity of the test in detecting Gram-negative bacteria was 63%, while the specificity was 97%. The predictive value of a positive test result was 70%, the figure being somewhat higher (75%) when the material was limited to milk samples without antibiotic residues. The predictive value of a negative test result was 95%. The LAL-test is considered to constitute a valuable cow-side test for the veterinary practitioner, aiding the selection of antibacterial drug of choice for the initial treatment of clinical mastitis.

Endotoxin and arachidonic acid metabolites in portal, hepatic and arterial blood of cattle with acute ruminal acidosis.

Ruminal acidosis was induced experimentally with 70 g barley/kg body weight in 2 rumen fistulated cows with chronic indwelling catheters in the portal vein, in a hepatic vein and the carotid artery. The cows were followed for 24 and 20h after the overfeeding and evaluated clinically and by clinical chemistry. The 2 cows exerted different responses to the treatment. Both cows showed signs of severe ruminal acidosis. Both cows had endotoxin in portal and hepatic vein blood, but only 1 of the cows convincingly developed a systemic endotoxaemia. A pre-hepatic release of the stable prostacyclin and thromboxane metabolites, 6-ketoprostaglandin F1 alpha and thromboxane B2 was demonstrated in this cow. The results of the present study show that endotoxin and arachidonic acid metabolites of pre-hepatic origin may be factors involved in the pathogenesis of ruminal acidosis, and that investigation of the factors affecting translocation of ruminal endotoxin and subsequent clearing in the liver, will be of importance.

Endotoxin levels in milk and plasma of mastitis-affected cows measured with a chromogenic limulus test.

A chromogenic limulus test ("Toxicolor") was applied to cow's milk and plasma after treatment with perchloric acid to remove interfering factors. The endotoxin levels in normal cow's milk and plasma were all less than 10 pg ml-1. In acute mastitis, the milk endotoxin level averaged (1.1 +/- 0.7) X 10(3) pg ml-1 in the cases where Gram-negative bacteria were isolated, while the plasma endotoxin concentration was normal. The endotoxin levels in the quarters infected with Gram-positive bacteria were all normal, both in milk and plasma. In gangrenous mastitis due to Gram-negative bacteria, the endotoxin concentration was very high in both milk [(9.3 +/- 5.3) X 10(6) pg ml-1] and plasma (85.2 +/- 68.2 pg ml-1). In similar cases due to Gram-positive bacteria, endotoxin levels were all normal, both in milk and plasma, resembling the acute mastitis due to Gram-positive bacteria. The test was considered suitable for the diagnosis of mastitis due to Gram-negative organisms and the levels of endotoxin detected would aid in assessing the prognosis.

Plasma endotoxin concentrations in experimental and clinical equine subjects.

Endotoxin (LPS) was quantitated in experimental subjects and in horses with naturally occurring gastrointestinal strangulation obstruction and/or septicaemic diseases to establish the fate of LPS and the clinical usefulness of the Limulus amoebocyte lysate (LAL) assay. The assay was validated for sensitivity (10 pg/ml), recovery (90 to 106 per cent), intra-assay precision (CV = 5.5 per cent) inter-assay precision (CV = 11 per cent), and stability of diluted, heat treated, frozen samples (at least 90 days). Plasma concentrations of LPS after sublethal (3 micrograms/kg) jugular or portal vein bolus injections of LPS rose to 4000 pg/ml and 1500 pg/ml respectively followed by a rapid phase of clearance. Peak plasma concentrations of LPS, associated with slow portal infusion, were lower than peak values associated with bolus injections, remained elevated during the infusion (2 h), but rapidly decreased after infusion was stopped. Thirty seven horses with 38 episodes of naturally occurring gastrointestinal or septicaemic disease were assayed for LPS. Eight episodes involving gastrointestinal disease and eight involving septicaemic disease were positive for LPS. It is concluded that the LAL assay is sensitive and reliable for detecting LPS in equine plasma and it may have clinical value for establishing the severity of endotoxaemia or for distinguishing between septic and non-septic conditions. Problems of rapid clearance of LPS from plasma, low concentrations, the possibility of sample contamination, and the time and method of sample procurement remain to be addressed.

Detection of endotoxin in cases of equine colic.

The Limulus amoebocyte lysate assay was used to test for the presence of endotoxin in 37 clinical cases of equine colic. Positive plasma titres were detected in 10 cases and the presence of endotoxin was significantly correlated with a high heart rate, a high packed cell volume and a poor prognosis. High levels of endotoxin were detected in gut contents taken from several sites in the gastrointestinal tract of normal horses.

Plasma endotoxin and concentrations of stable metabolites of prostacyclin, thromboxane A2, and prostaglandin E2 in postpartum dairy cows.

The presence of endotoxin in plasma and patterns of stable metabolites of prostacyclin (PC), thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) were determined during the first postpartum estrous cycles in sixteen dairy cows. These included 8 cows with uterine infections which exhibited shortened luteal phases (SC) and 8 cows which had normal luteal phases (NC) after the first post partum ovulations. Endotoxin was consistently detected in all SC cows during the abbreviated estrous cycles while plasma samples of NC cows were free of endotoxin. Plasma concentrations of TXA2 metabolite was higher in SC cows (p less than 0.05) (1785-3452 pg/ml) compared to NC cows (723-1240 pg/ml). Similarly, plasma concentrations of PC metabolite was higher in SC cows (p less than 0.07) (423-1847 pg/ml) compared to NC cows (159-325 pg/ml). In contrast, plasma concentrations of PGE2 metabolite was higher in NC cows (p less than 0.05) (850-2219 pg/ml) compared to SC cows (455-628 pg/ml). The results of this study suggest that postpartum uterine infections mediate the release of prostaglandins from the uteri by means of the endotoxin and endotoxin appears to stimulate selectively the production of PC and TXA2 favoring early demise of corpora lutea formed after first postpartum ovulations in dairy cows.

Comparison of a gelation and a chromogenic Limulus (LAL) assay for the detection of gram-negative bacteria, and the application of the latter assay to milk.

When a chromogenic Limulus Amoebocyte Lysate (LAL) assay and a tube gelation LAL assay were compared for the detection of Gram-negative bacteria using a strain of Pseudomonas putida, the detection level (approximately 10(3) cfu/ml) and cost of the assays were approximately the same for both assays but the reading was more precise for the chromogenic substrate assay. A modified chromogenic assay was devised for detection of Ps. putida in milk.

[Limulus test--a rapid and simple method for the detection of endotoxins produced by gram-negative bacteria in mastitis milk]. / Limulustestet--en snabb och enkel metod att påvisa endotoxin från gramnegativa bakterier i mjölk vid mastit.

According to the present study the limulus amebocyte lysate test (LAL) seems to be a convenient test to detect endotoxin in milk from udder quarters with and without inflammation. The correlation between endotoxin concentration and the results from the bacteriological investigation of 79 milk samples was good (Table I). Determination of endotoxin in 20 milk samples from cases of acute clinical mastitis with high cell count and a negative bacteriological culture showed that all but one had an endotoxin concentration of greater than 1.0 ng/ml milk (Table II). By using a micromethod of the LAL it is possible to detect cases of mastitis caused by gram-negative bacteria about one hour after the sample has reached the laboratory. In a preliminary field study milk from 13 cases of acute clinical mastitis were tested by a modified limulus amebocyte lysate (LAL) test ("cowshed test"). A 100% correlation to bacteriological findings was observed (Table IV). By using the LAL test to detect mastitis cases caused by gram-negative bacteria great economic advantages and less risk for resistance problems can be achieved by using proper antibiotics. This is the fact in Sweden where the frequency of acute clinical mastitis caused by streptococci (100% of strains sensitive for penicillin) and Staphylococcus aureus (about 90% of strains sensitive for penicillin) is high (70-80%) and about 20% are caused by gram-negative bacteria, mostly E. coli.