Nitric oxide synthesis is depressed in Bos indicus cattle infected with Trypanosoma congolense and Trypanosoma vivax and does not mediate T-cell suppression

Infection with African trypanosomes causes the diseases sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. Suppression of cellular immune responses is a feature of trypanosomiasis in bovine, human, and murine hosts. Some aspects of immunosuppression in the murine model are mediated by nitric oxide (NO) produced by gamma interferon (IFN-gamma)-activated macrophages. We have investigated whether a similar mechanism is responsible for T-cell unresponsiveness in bovine trypanosomiasis. Bovine monocytes and macrophages from uninfected cattle and activated in vitro with IFN-gamma produced NO; however, this response was down-regulated in infected cattle. Similarly, the expression of inducible NO synthase messenger RNA was depressed in macrophages of infected cattle. Proliferation of mononuclear cells of trypanosome-infected cattle cultured with mitogen or trypanosome antigens was unchanged by the addition of an NO synthase inhibitor. Lymphocytes of infected cattle secreted interleukins with T-cell growth factor activity after in vitro activation with mitogens but not after activation with trypanosome antigens. Although lymph node cells secreted IFN-gamma after in vitro activation, ex vivo expression of mRNA was depressed. In contrast, the level of expression of interleukin 10 mRNA was higher during infection. We conclude that NO is not involved in the loss of T-cell proliferative function associated with trypanosomiasis in cattle and that, in contrast to the mouse model, the capacity of monocytes and macrophages to produce NO is actually down-regulated in infected cattle.     

Study on the sequential tsetse-transmitted Trypanosoma congolense, T. brucei brucei and T. vivax infections to African buffalo, eland, waterbuck, N'Dama and Boran cattle

Susceptibility of African buffalo, eland, waterbuck, N'Dama and Boran cattle to sequential Glossina morsitans centralis-transmitted infections of Trypanosoma congolense, T. brucei brucei and T. vivax was compared, and their possible role as reservoirs of these parasites for G. moristans centralis, G. pallidipes, G. austeni, G. brevipalpis and G. longipennis determined. The buffalo, eland, waterbuck and N'Dama controlled T. congolense parasitaemias and were able to prevent anaemia. By contrast, one Boran became severely anaemic whilst the other controlled parasitaemia and anaemia. When the above five species of Bovidae were rechallenged with T. brucei brucei they showed persistent parasitaemias but did not develop anaemia. The buffalo died of other causes. When the remaining four bovids were rechallenged with T. vivax they became infected with mixed T. vivax/T. b. brucei parasites. Eland, waterbuck and N'Dama controlled parasitaemias and anaemia whereas the Boran became anaemic. Cyclical development of T. congolense occurred in G. moristans centralis when fed on the bovid hosts, with buffalo being infective for tsetse flies for a much longer period. There was no relationship between the levels of T. congolense parasitaemia in the bovid hosts and the resultant infection rates in tsetse flies. Glossina m. centralis was more susceptible than G. pallidipes to T. brucei brucei whilst G. austeni the least; G. brevipalpis and G. longipennis were refractory to the mature infection. Again there was no relationship between T. brucei brucei parasitaemia levels in the hosts and infection rates in the flies. Glossina m. centralis and G. pallidipes showed mixed T. brucei brucei/T. vivax infections whilst G. austeni, G. brevipalpis and G. longipennis became infected with T. vivax alone. Tsetse flies showed higher T. vivax infection rates when fed on the hosts with high parasitaemias than thosewith low parasitaemias. Thus trypanotolerant African buffalo, eland, waterbuck, N'Dama as well as some trypanosusceptible Boran cattle can serve as reservoirs of single or mixed trypanosome infections for tsetse flies. This study has also shown that the Ag-ELISA on the sera from the five bovid hosts had low sensitivity and species-specificity. Examinations of thin wet blood films and buffy coats with a phase-contrast microscope were not sensitive enough to detect the parasites on all occasions. Xenodiagnosis using mice for T. brucei brucei and T. congolense infections, and tsetse flies for all the three trypanosome species were most sensitive for the detection of trypanosome infections in the bovid hosts.     

Outbreaks of trypanosomosis due to Trypanosoma vivax in cattle in Bolivia

Some new commercial methods for the extraction of viral RNA have been introduced recently. In addition to the study published previously (Verhofstede, C., Reniers, S., Van Wanzeele. F., Plum J., 1996. AIDS 8, 1421-1427), seven different methods (four newly developed and three reference methods) for extraction of HIV-1 RNA from plasma have been evaluated. The RNA preparation method that gave the best results (acceptable reproducibility, highest sensitivity, reasonable price, fast and easy to perform), was the QIAamp Viral RNA kit from QIAgen. The High Pure Viral RNA Kit (Boehringer Mannheim) as well as the non-commercialised extraction kits were also very sensitive. The non-commercial tests seem less suitable for routine use and for the processing of large number of samples. Two methods, RNA Insta-Pure LS (Eurogentec) and PANext RNA extraction kit 1 (NTL, PANsystems GmbH) are not adapted for HIV plasma extraction. The single step methods using glass fibre or silica column are rapid (from 60 to 75 min depending on the number of wash steps) and although the price is high they are cheaper than the Boom extraction methods: High Pure Viral RNA Kit (Boehringer Mannheim) ($3.3/sample), QIAamp Viral RNA Kit (Qiagen) ($3.6/sample), Boom extraction ($5/sample). The Qiagen kit is the only kit that combines sensitivity with reproducibility, it is commercialised, rapid and affordable in price and can be automated. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extractions varied between 26.4 and 48.6%).     

Extravascular localization of Trypanosoma vivax in cattle (author''s transl)

In 2 cattle experimentally infected with Trypanosoma vivax, strain EATRO 1695, trypanosomes were found in the marginal sinus of one of several lymph nodes examined. One animal showed trypanosomes in a small focus in the interstitial tissue of the heart. It is concluded that Trypanosoma vivax can not be regarded as a strict plasma parasite.     

Sensitivity and specificity of dipstick tests for rapid diagnosis of malaria in nonimmune travelers

Swift diagnosis of Plasmodium falciparum malaria in areas where the disease is not endemic is frequently complicated by the lack of experience on the side of involved laboratory personal. Diagnostic tools based on the dipstick principle for the detection of plasmodial histidine-rich protein 2 (HRP-2) and parasite-specific lactate dehydrogenase (pLDH), respectively, have become available for the qualitative detection of P. falciparum malaria. In order to evaluate two of the currently available assays, specimens from 231 patients were screened during a prospective multicenter study. Among the screened specimens, samples from 53 patients (22.9%) were positive for P. falciparum malaria by microscopy and/or PCR. While the test kit based on the detection of HRP-2 performed with a sensitivity of 92.5% and a specificity of 98.3%, the kit for the detection of pLDH showed a sensitivity of 88.5% and a specificity of 99.4%. Dipstick tests have the potential of enhancing speed and accuracy of the diagnosis of P. falciparum malaria, especially if nonspecialized laboratories are involved.     

Derivation and characterization of a quinapyramine-resistant clone of Trypanosoma congolense

Over a period of 208 days a quinapyramine-resistant population was derived in vivo from a quinapyramine-susceptible clone of Trypanosoma congolense: IL 1180. While the dose of quinapyramine sulfate required to cure 50% of mice infected with the parental clone was 0.23 mg/kg of body weight, the 50% curative dose for the resistant derivative, IL 1180/Stabilate 12, was greater than 9.6 mg/kg. This approximately 40-fold increase in resistance to quinapyramine was shown to be associated with an 8-fold increase in resistance to isometamidium, a 28-fold increase in resistance to homidium, and a 5.5-fold increase in resistance to diminazene. Cross-resistance to homidium and diminazene was also demonstrated in goats. Two clones derived from the drug-resistant derivative underwent cyclical development in Glossina morsitans centralis, producing mature infection rates of 39.6 and 23.9%. Thus, induction of resistance to quinapyramine in T. congolense IL 1180 was associated with cross-resistance to isometamidium, homidium, and diminazene and did not compromise the population's ability to undergo full cyclical development in tsetse flies.     

Rise in erythropoietin concentrations in experimental Trypanosoma congolense infection of calves

A bioassay was used to measure erythropoietin (EPO) concentrations in calves with haemorrhagic anaemia due to blood loss and in calves with anaemia due to Trypanosoma congolense infection. The bioactivity of EPO was measured in the assay by its stimulatory effect on 125I-deoxyuridine incorporation in spleen cells from phenylhydrazine-treated mice. Erythropoietin concentrations in blood-volume-depleted calves were elevated 6 h after blood loss, maximal (1225 mU/ml) at 33 h and below detection limits at 72 h. Reticulocytes (0.05 +/- 0.1%) appeared in blood by 72 h, peaked at 120 h and disappeared from the circulation by 7 days after bleeding. The packed cell volume (PCV) started increasing at 120 h and reached near pre-bleeding values by 14 days. In T. congolense-infected calves, parasites were first detected in the peripheral blood 12 days post-infection (dpi). Parasitaemia peaked (5 x 10(5) trypanosomes/ml of blood) at 15-18 dpi and, thereafter, several waves of parasitaemia were observed, but the peaks gradually diminished. Undiluted plasma from T. congolense-infected calves suppressed 125I-deoxyuridine incorporation into spleen cells from 13 dpi onwards. The suppressive effect of plasma was partly negated by five-fold dilution, which made possible the detection of increased EPO concentrations during the acute and chronic stages of the anaemia. The highest EPO peaks, reaching 2300 mU/ml in one calf, were detected during the chronic stage of the infection. At 15-39 dpi, there was a transient bone-marrow erythropoietic response characterized by an increase in mean corpuscular volume and a decrease in mean corpuscular haemoglobin concentration but with few reticulocytes (0.4%). However, from 76 dpi onwards, this response waned despite low PCV and elevated EPO concentrations. These results suggest that there is an ineffective erythroid response in the face of elevated EPO concentrations during bovine trypanosomiasis. The negative effect of plasma and serum from trypanosome-infected calves on the in-vitro bioactivity of EPO suggests the presence of inhibitory factors.     

Sensitivity and specificity of a rapid whole-blood assay for D-dimer in the diagnosis of pulmonary embolism

BACKGROUND: Patients with suspected pulmonary embolism often have nondiagnostic lung scans and may present in circumstances where lung scanning is unavailable. Levels of D-dimer, a fibrin-specific product, are increased in patients with acute thrombosis; this may simplify the diagnosis of pulmonary embolism. OBJECTIVE: To determine the sensitivity and specificity of a whole-blood D-dimer assay in patients with suspected pulmonary embolism and in subgroups of patients with low pretest probability of pulmonary embolism or nondiagnostic lung scans. DESIGN: Prospective cohort. SETTING: Four tertiary care hospitals. PATIENTS: 1177 consecutive patients with suspected pulmonary embolism. MEASUREMENTS: All patients underwent an assessment of pretest probability by use of a standardized clinical model, a D-dimer assay, ventilation-perfusion lung scanning, and bilateral compression ultrasonography. Patients in whom pulmonary embolism was not initially diagnosed were followed for 3 months. Accordingly, patients were categorized as positive or negative for pulmonary embolism. RESULTS: Of the 1177 patients, 197 (17%) were classified as positive for pulmonary embolism. Overall, the D-dimer assay showed a sensitivity of 84.8% and a specificity of 68.4%. In 703 patients (3.4%) with a low pretest probability of pulmonary embolism, the likelihood ratio associated with a negative D-dimer test result was 0.27, resulting in a posterior probability of 1.0% (95% CI, 0.3% to 2.2%). In 698 patients with nondiagnostic lung scans (previous probability, 7.4%), the likelihood ratio associated with a negative D-dimer test result was 0.36, resulting in a posterior probability of 2.8% (CI, 1.4% to 4.8%). CONCLUSIONS: A normal D-dimer test result is useful in excluding pulmonary embolism in patients with a low pretest probability of pulmonary embolism or a nondiagnostic lung scan.     

Time-dose response of Trypanosoma congolense bloodstream forms to diminazene and isometamidium

Trypanosoma congolense bloodstream forms were propagated in vitro axenically in a simplified cultivation medium at 34 degrees C. Viability of a drug-sensitive and a drug-resistant clone were examined for 10 days following exposure to 0.1, 1.0 and 10.0 micrograms ml-1 of diminazene aceturate and 0.1, 1.0 and 10.0 ng ml-1 of isometamidium chloride for various time intervals. Drug-sensitive T. congolense were irreversibly damaged after incubation with 10 micrograms ml-1 or 1 microgram ml-1 diminazene aceturate for 30 min or 2 h, respectively, while drug-resistant trypanosomes were not affected. Exposure to 10 ng ml-1 isometamidium chloride eliminated drug-sensitive trypanosomes after 24 h and drug-resistant trypanosomes after 96 h. The data obtained on in vitro time-dose responses of T. congolense were related to pharmacokinetic data of diminazene and isometamidium in cattle plasma.     

Experimental murine Trypanosoma congolense infections. I. Administration of anti-IFN-gamma antibodies alters trypanosome-susceptible mice to a resistant-like phenotype

The mechanisms regulating resistance or susceptibility to African trypanosomes have been enigmatic. In this study, we assessed the production of several cytokines (IL-4, IFN-gamma, and TNF-alpha) in vivo and in vitro using genetically susceptible (BALB/c) or resistant (C57BL/6) mice infected with cloned Trypanosoma congolense and the role of these cytokines in pathogenesis of this infection. Plasma of infected BALB/c mice contained higher levels of IL-4 and IFN-gamma than the plasma of infected C57BL/6 mice. Conversely, plasma TNF-alpha levels were elevated significantly in the resistant mice relative to the susceptible ones. Splenic IFN-gamma mRNA appeared earlier and were maintained at higher levels in infected BALB/c than in C57BL/6 mice. Both spontaneous and Con A-induced secretions of IL-4 and IFN-gamma by splenocytes from infected BALB/c mice were significantly higher than those from their C57BL/6 counterparts. Con A-induced proliferation of splenocytes from infected BALB/c mice was progressively suppressed. Nitric oxide was not involved in this suppression, but the suppression was positively correlated with IFN-gamma secretion. Addition of neutralizing Abs to IFN-gamma to cultures of Con A-stimulated spleen cells from infected BALB/c mice effectively reversed this suppression. Furthermore, administration of anti-IFN-gamma Abs to BALB/c mice early during infection dramatically shifted the phenotype of these susceptible mice to a more resistant-like phenotype, as expressed by a low and undulating parasitemia and a >300% increase in survival period. These results strongly suggest that the enhanced induction and secretion of IFN-gamma during T. congolense infections contribute to the relative susceptibility of BALB/c mice to the disease.     

Acquisition of resistance to the tick Amblyomma variegatum in Boran cattle, Bos indicus and the effects of Trypanosoma congolense and Babesia bigemina on host resistance

Resistance was induced in cattle to the tick Amblyomma variegatum by five consecutive infestations with nymphs and adults. Using the principal component analysis (PCA), it was found that percentage of adults engorged, percentage of adults which died, percentage of nymphs which engorged, percentage of nymphs which moulted and percentage of nymphs which died, were the main indicators of resistance against A. variegatum. The percentages of nymphs which engorged or moulted after the third infestation were significantly (P < 0.01) reduced while the percentage of nymphs which died increased significantly (P < 0.01) after the third infestation. Percentages of adults which engorged or died started to decrease significantly (P < 0.01) from the fourth infestation after an initial increase during this period. The acquisition of resistance by cattle to the adult ticks was slower than to the nymphs. Infection of cattle with Trypanosoma congolense and Babesia bigemina after the fifth infestation enhanced the acquired immunity as revealed by the significantly (P < 0.01) increased feeding period of the adult ticks and changes in other parameters.     

Plasmodium vivax infections in chimpanzees for sporozoite challenge studies in monkeys

The development and testing of vaccines directed against Plasmodium vivax has relied on Saimiri and Aotus monkeys as the animal test system and on chimpanzees to provide infective gametocytes to produce sporozoites for monkey challenge studies and vaccine development. One sporozoite-induced and 29 blood-induced infections with the Salvador I strain of P. vivax were studied in splenectomized chimpanzees. Eighteen primary infections with P. vivax resulted in maximum parasite counts ranging from 1,519 to 81,810/ microliters (median 29,100/microliters). Twelve infections induced in animals previously infected with the homologous or heterologous strains of P. vivax had maximum parasite counts ranging from 155 to 14,136/microliters (median 1,736/microliters). A total of 202 of 237 lots containing a total of 293,175 Anopheles freeborni, An. stephensi, An. gambiae, An. dirus, An. quadrimaculatus, and An. maculatus mosquitoes were infected by membrane feeding on gametocytes from chimpanzees. Despite lower levels of parasitemia during secondary (reinfection) parasitemia, 66 of 70 lots of mosquitoes (94.3%) were infected. Based on the mean number of oocysts per positive mosquito gut, An. freeborni was more heavily infected than An. stephensi; An. stephensi was more heavily infected than An. gambiae; there was no significant difference between An. stephensi and An. dirus. Sporozoites from An. stephensi, An. gambiae, An. dirus, and An. freeborni infected with the Salvador I strain of P. vivax produced in chimpanzees were used to infect 193 Saimiri and six Aotus monkeys as well as one chimpanzee.     

Sensitivity and specificity of mu-capture ELISA for detection of enterovirus IgM

The effect of Taxol on the radiation sensitivity of human squamous carcinoma of the head and neck region was determined in vitro, using clonogenic assays and multicellular tumor spheroids (MTS). Radiosensitivity parameters were determined by alpha and beta for clonogenic assays, and by the residual/control volume ratios at 2 Gy (RSV2) and the dose inducing 50% decrease in MTS number (SCD50) for spheroids. In HTB43 and CAL27 colonies, the combination was antagonist. In spheroids, Taxol induced a decrease of RSV2 and SCD50 in HTB43 and CAL27 MTS and their combinations with radiation were synergistic and additive, respectively. Therefore, the different results obtained by clonogenic assays and MTS may suggest higher drug incorporation through the multiple cell layers of the spheroids than in monolayers.     

The influence of energy intake on the pathophysiology of Trypanosoma congolense infection in Scottish blackface sheep

The intensity of parasitaemia, degree of anaemia, live body weight gains and blood biochemical changes were measured in two groups of Scottish Blackface sheep infected experimentally with Trypanosoma congolense and allowed either a high (9.9 MJ metabolisable energy (ME) day-1) or a low (6.1 MJ ME day-1) energy intake. It was observed that infected animals on the low energy intake had a longer mean prepatent period, but following patency they developed more severe anaemia and greater growth retardation than those on the high energy intake. Both infected groups exhibited significant reductions in serum total lipids, phospholipids, plasma cholesterol and albumin. However, these changes were more severe in the animals on the low energy intake than in those on the high energy intake. It was concluded that adequate energy nutrition enhances the ability of infected animals to withstand the adverse effects of infection, by promoting body weight gains and moderating the severity of the pathophysiological changes associated with ovine trypanosomosis.     

Sensitivity and specificity of serological and bacteriological tests for contagious bovine pleuropneumonia

In 1990 an outbreak of contagious bovine pleuropneumonia (CBPP) occurred in Italy. Subsequent surveillance for CBPP was based on random sampling in bovine herds, serological controls on all animals moved from the herd of origin and controls on slaughtered animals. Official tests employed were the complement fixation test (CFT) and bacteriological isolation and typing. A total of 33,856 serum samples collected from herds in CBPP-free regions were used to define CFT specificity, while samples from 595 animals from infected herds were employed to define the sensitivity. Ninety-nine animals from three infected herds were used to estimate the sensitivity of the isolation technique. Results showed the specificity of CFT (threshold +1:10) to be 98% and sensitivity to be 63.79%. The sensitivity of the test did not change significantly, regardless of whether the lesions were caused by acute or chronic infection. The sensitivity of the isolation technique was 54.1%.     

Variation in sensitivity of Trypanosoma congolense to diminazene during the early phase of tsetse-transmitted infection in goats

Twenty-five goats were randomly allocated to five groups of five animals each and infected with Trypanosoma congolense IL 3274 via the bites of infected Glossina morsitans centralis. At intervals of 1, 4, 8, 12 or 19 days following infection, each group of five animals was treated intramuscularly with diminazene aceturate at a dose of 7.0 mg kg-1 body weight (b.w.). While treatment on Day 1 eliminated infections in all five goats, treatment on Day 19 did not cure any of the animals; in groups treated 4, 8 or 12 days following infection, two of five goats in each group were cured. Since the alteration in apparent resistance of T. congolense IL 3274 between Day 1 and Day 19 could have been due to alteration in expression of drug resistance by trypanosomes as the population expanded, the experiment was repeated using trypanosomes that reappeared in the animals that had been treated with diminazene aceturate on Day 19. On Day 36, when all five animals were parasitaemic, five groups of teneral G. m. centralis, each containing 160 flies, were fed on one occasion on each of the five goats (one group of testse flies per goat). Thereafter, each group of tsetse flies was maintained on clean rabbits. When infective, five flies from each group were allowed to feed on two naive goats each (i.e. two goats per group of tsetse flies). One animal in each pair was treated 24 h after infection with diminazene aceturate at a dose of 7.0 mg kg-1 b.w., the other was treated on Day 19, when parasitaemic, with the same drug dosage. As before, treatment 24 h following infection eliminated infections in all animals, but when treatment was delayed until Day 19, trypanosomes in all animals were refractory to treatment. Thus, although tsetse flies were infected with trypanosomes that had arisen in infected goats following treatment with diminazene aceturate at a dose of 7.0 mg kg-1 b.w., when the same flies were allowed to feed on clean goats, the resultant infections were sensitive to treatment with the same drug dosage when administered 24 h following infection. These data therefore indicate that there is a significant alteration in diminazene sensitivity of IL 3274 between Day 1 and Day 19 and that this is associated with an alteration in the resistance phenotype of the trypanosomes.     

Monoclonal antibody-based ELISAs for part-per-billion determination of polycyclic aromatic hydrocarbons: effects of haptens and formats on sensitivity and specificity

BACKGROUND: Mitral annuloplasty is an important element of most mitral repairs, yet the effects of various types of annuloplasty rings on mitral annular dynamics are still debated. Recent studies suggest that flexible rings preserve physiologic mitral annular area change during the cardiac cycle, while rigid rings do not. METHODS: To clarify the effects of mitral ring annuloplasty on mitral annular dynamic geometry, we sutured 8 radiopaque markers equidistantly around the mitral anulus in 3 groups of sheep (n = 7 each: no ring, Carpentier-Edwards semi-rigid Physio-Ring [Baxter Healthcare Corp, Edwards Division, Santa Ana, Calif], and Duran flexible ring [Medtronic, Inc, Minneapolis, Minn]). Ring sizes were selected according to anterior leaflet area and inter-trigonal distance (Physio-Ring 28 mm, n = 7; Duran ring 31 mm, n = 5, and 29 mm, n = 2). After 8 +/- 1 days of recovery, the sheep were sedated and studied by means of biplane videofluoroscopy. Mitral annular area was calculated from 3-dimensional marker coordinates without assuming circular or planar geometry. RESULTS: In the no ring group, mitral annular area varied during the cardiac cycle by 11% +/- 2% (mean +/- SEM; maximum = 7.6 +/- 0.2, minimum = 6.8 +/- 0.2 cm2; P 相似文献