Identification of Dirofilaria immitis proteins recognized by antibodies from infected dogs.

The identification of excreted-secreted (ES) proteins of filarial nematodes as potential diagnostic reagents is an important requirement for the development of methods to determine level of infection in the host, especially for human filariae. Dirofilaria immitis, the canine heartworm, is a widespread and important veterinary pathogen and is a useful model for filarial parasites of humans. An analysis of proteins released from adult D. immitis (the secretome) in culture is available. We sought to identify D. immitis ES proteins found in vivo to validate the in vitro secretome and to investigate them as potential diagnostic reagents. Cultures of D. immitis adults obtained from infected dogs were maintained for 72 hr with daily changes of media. Proteins were concentrated from spent media by standard methods and were passed through Protein-A columns containing purified IgG antibodies from heartworm-infected dogs. Following extensive washing, heartworm proteins recognized by the antibodies were eluted from these columns and submitted for analysis by tandem mass spectrometry (MS/MS). As a comparison, somatic proteins from adult D. immitis female parasites and microfilaria were also processed and analyzed by the same protocol. Six, 9, and 12 proteins were identified by MS/MS in the ES, adult female, and microfilaria samples, respectively. The identification of the most abundant parasite proteins present in the serum of infected hosts offers a rational approach to the development of new diagnostic assays that may be applicable across the Filarioidea.

The association of adult Onchocerca volvulus with lymphatic vessels.

Immunocytochemical examination of onchocercal nodule tissues containing adult Onchocerca volvulus using immuno-markers for blood and lymphatic vessels (vWF, D2-40, podoplanin, Prox-1, and Lyve1) shows a distinct pattern of distribution of these vessels within nodules. Blood vessels were commonly seen associated with organized lymphoid cellular aggregates in the both the outer and inner areas of the nodules. In contrast, the majority of the lymphatic vessel positivity was seen in the central zone in close apposition to the adult parasites, and the remainder usually associated with microfilariae in the outer areas of the nodule. These findings suggest an intimate relationship between adult O. volvulus and lymphatic vessels, including the likely proliferation of lymphatic endothelial cells (lymphangectasia) akin to that seen with other filariae. These findings indicate that adult O. volvulus may migrate via the lymphatic system, and that clinical manifestations of this disease that involve tissue edema may be the result of the location of these worms in the lymphatic system.

Lymphatic filariasis: patients and the global elimination programme.

The defining images of lymphatic filariasis are the horrendous disfigurements of lymphoedema, elephantiasis and hydrocele. These clinical presentations, although obviously important and life changing, are not, however, the only outcomes of this wide-spread filarial infection. The other effects of the disease range from severe, acute but short-term bouts of sickness to psychological impairment, poverty and family hardship. It is important to support cases of the disease through all means available, such as reparative hydrocelectomy, hygiene training and facilitation, and the provision of adequate chemotherapy. Although only a minority of the residents in any endemic community is affected with the severe clinical manifestations of this parasitic infection, these cases are central to, and important advocates for, the current global effort to eliminate the infection through mass drug administrations (MDA). Their clinical improvement acts as an important catalyst for the general population and encourages high compliance in the MDA. This communication discusses the central role that filariasis patients have played in the Tanzania Lymphatic Filariasis Elimination Programme to date, and covers some of the clinical successes achieved in the past 10 years. The abolition of the clinical manifestations of filarial infection remains the ultimate goal of the Global Programme to Eliminate Lymphatic Filariasis, and maintaining a focus on the affected individuals and their clinical condition is vital to that programme's overall success.

The sharp end - experiences from the Tanzanian programme for the elimination of lymphatic filariasis: notes from the end of the road.

The Tanzania Lymphatic Filariasis Programme, which was launched in 2000, is, in terms of geographical coverage, among the largest disease-control programmes in Tanzania's history, currently reaching 9.4 million people in 34 districts. The issues associated with this programme's implementation are reviewed here, in the context of the various players/stakeholders involved. This article provides an insight of how the programme began and discusses key areas in the programme's design. Mainly, however, it gives some impressions of how the programme is perceived by, and how it affects, village healthworkers, patients and politicians - the people who contribute to the implementation of the programme at various levels.

Endotoxin contamination in the dental surgery.

Dental waterlines contain large numbers of Gram-negative bacteria. Endotoxin, a component of such organisms, has significant health implications. Paired samples of dental unit water and the aerosols generated during dental procedures were collected, and assayed for bacteria and endotoxin levels, using heterotrophic plate counts and the Limulus amoebocyte lysate test. Consistent with published studies, the extent of bacterial contamination in the dental waters sampled for this investigation surpassed the levels associated with potable water, with counts in excess of 2.0x10(6) c.f.u. ml(-1) in some samples. Correspondingly high concentrations of endotoxin [up to 15 000 endotoxin units (EU) ml(-1)] were present in the water. A statistically significant Spearman correlation coefficient of rho=0.94 between endotoxin (EU ml(-1)) and bacterial load (c.f.u. ml(-1)) was demonstrated. All of the aerosol samples contained detectable endotoxin. Further studies of the consequences of dental endotoxin exposure, and evaluation of means to prevent exposure, are warranted.

Quinine therapy in severe Plasmodium falciparum malaria during pregnancy in Sudan.

A prospective study was carried out in an area of unstable malaria transmission in central Sudan to determine the efficacy and toxicity of quinine in pregnancy. Thirty-three pregnant women with severe Plasmodium falciparum malaria at mean 28.8 weeks gestational age were treated with quinine for 7 days. The mean body temperature on presentation for 3 patients who delivered prematurely was significantly higher than for those who delivered at term (39.2 +/- 0.7 degrees C versus 38.7 +/- 1.3 degrees C). There were no significant difference between the 2 groups in other clinical or biochemical parameters. There were no clinically detectable congenital malformations and no auditory, visual or other neurological deficits in the babies at birth or 6 months later. Quinine may be safe in the treatment of severe falciparum malaria during pregnancy.

Quinine therapy in severe Plasmodium falciparum malaria during pregnancy in Sudan

A prospective study was carried out in an area of unstable malaria transmission in central Sudan to determine the efficacy and toxicity of quinine in pregnancy. Thirty-three pregnant women with severe Plasmodium falciparum malaria at mean 28.8 weeks gestational age were treated with quinine for 7 days. The mean body temperature on presentation for 3 patients who delivered prematurely was significantly higher than for those who delivered at term [39.2 +/- 0.7 degrees C versus 38.7 +/- 1.3 degrees C]. There were no significant difference between the 2 groups in other clinical or biochemical parameters. There were no clinically detectable congenital malformations and no auditory, visual or other neurological deficits in the babies at birth or 6 months later. Quinine may be safe in the treatment of severe falciparum malaria during pregnancy

Bombesin-like neuropeptides in nematodes.

This paper reports evidence of members of the bombesin-like peptide family in a number of nematodes, including Caenorhabditis, Panagrellus, Dirofilaria, Onchocerca, Brugia, Haemonchus, Ostertagia, Toxocara, and Ascaris. One of these (Ostertagia) secretes the bombesin-like material into its environment. Specific binding of gastrin-releasing peptide to the hypodermis consistent with the presence of receptors was demonstrated. These data suggest that this class of peptides may play an important role in nematode hypodermal physiology.

Patellar tendon augmentation after removal of its central third limits joints tissue changes.

The central third of the patellar tendon is commonly used to reconstruct the injured anterior cruciate ligament. Some studies have noted changes in joint tissues following this procedure. It has been postulated that these changes may be associated with increased stress on the remaining tendon following harvest of the graft. In our study, the central third of the patellar tendon was excised in three groups of rabbits. The central tendon defects in two of the three groups were fitted with different augmentation devices to augment the host tendon during the healing process. All rabbits followed a daily treadmill exercise regimen for 12 weeks following the operation. Biomechanical testing of the tendon revealed that in nonaugmented tendons the cross-sectional area and the length of the patellar tendon significantly increased 112 and 16%, respectively. There was histological evidence of host-tendon remodeling throughout the cross section and extensive fibrosis in the infrapatellar fat pad. Augmentation of the tendon significantly reduced these changes, with the least change noted in the group with the greatest augmentation. The rabbits with augmentation devices retained tendon dimensions similar to those of the contralateral intact tendon, and tendon remodeling occurred only in the defect area. The rabbits with augmentation devices exhibited little to no fibrosis of the fat pad. Structural properties of augmented and nonaugmented tendons were similar despite the size differences, indicating higher tissue quality in the augmented tendons. This study suggested that complications of the knee joint (i.e., tendon proliferation and fat pad fibrosis) noted after anterior cruciate-ligament reconstruction with the autogenous patellar tendon may be limited by the implantation of an augmentation device.

Effect of octreotide on the pathology of hepatic schistosomiasis.

In clinical practice, octreotide (CAS 83150-76-9) has its greatest impact in the management of bleeding varices. The present work is the first one which was undertaken to investigate the possible use of octreotide as an antifibrotic agent and to study its effect on hepatic vasculature in Schistosoma mansoni infection. The material of this investigation consisted of two groups of albino mice (A, B), subdivided each into normal control, infected control, subgroups treated with octreotide, praziquantel (CAS 55268-74-1), and a combination of octreotide and praziquantel. Groups A and B were sacrificed at the 8th week and the 18th week post infection, respectively. By analysis of the obtained results, octreotide induced a reduction of the portal pressure, the weight of the spleen and the liver, the liver egg load (number of eggs) granuloma size and cellularity, and of the degree of hepatic fibrosis quantified by serum N-terminal peptide of type III procollagen in serum, serum laminin and tissue collagen using a Picrosirius red dye assay. Moreover, the biochemical state of hepatocytes has been improved. The subgroups treated with octreotide in association with praziquantel revealed better results than the subgroups treated with praziquantel alone. These obtained data were analysed in terms of histological extent of liver fibrosis in sections stained with Masson trichrome and sirius red, hepatocytic and sinusoidal changes at an ultrastructural level and by immunohistochemical demarcation of endothelial cells of blood vessels through the determination of factor VIII-related antigen. The promising results detected in this study may encourage to further investigate the positive findings of this drug with the intention of its possible application on a clinical level.

Blunt impact causes changes in bone and cartilage in a regularly exercised animal model.

The goal of this study was to document the effect of blunt-impact trauma on the knee in a small animal model that incorporated a known level of physical exercise after impact. We hypothesized that a single blunt impact to the patellofemoral joint, of a magnitude comparable with our earlier studies, would result in degenerative changes to cartilage and to subchondral bone of the patella. Blunt impacts were delivered to rabbit patellofemoral joints without producing bone fracture, and biomechanical and histological analyses were performed on joint tissues at various times. At 12 months, the subchondral bone plate was thicker on the impacted side than on the unimpacted side and than that of the controls at comparable locations (near where surface fissures were found on the impacted side). The instantaneous modulus of cartilage was significantly less on the impacted side than that of controls at 3, 6, and 12 months after impact. The relaxed modulus of cartilage on the unimpacted side increased with time after impact and was significantly greater than that of controls at 12 months. These facts suggest that in this exercise model, the contralateral limb should not be considered a control. The retropatellar cartilage on the impacted side was significantly less thick than that of controls at 12 months, and histological analyses of the cartilage and bone indicated early-stage osteoarthrosis in the impacted joint. Thus, in this animal model a single subfracture blunt impact produced degeneration of joint tissues.

Latent turkey herpesvirus infection in lymphoid, nervous, and feather tissues of chickens.

In earlier studies, we found that a late gene product, glycoprotein B (gB) was highly expressed in lymphoid tissues of chickens inoculated with turkey herpesvirus (HVT). The objectives of the present study were twofold. First, we wanted to expand on our previous research and determine if gB expression declines or disappears during later time periods of HVT infection. Second, we wanted to correlate gB expression with presence of HVT, i.e. if gB expression is absent, can HVT still be detected? Fifteen 1-day-old chicks were inoculated by intraperitoneal inoculation with 2000 plaque forming units of strain FC126 HVT. Thymus, spleen, bursa, brachial plexus, sciatic plexus, and feather tips were harvested at 21, 28, 35, 70, and 105 days postinoculation (PI). Brachial plexus and sciatic plexus were examined at 21, 28, and 35 days PI, and feather tips were examined at 21 and 28 days PI. An indirect immunofluorescence assay was used to detect HVT gB expression, and an in situ hybridization assay was used to detect HVT. At 21 days PI, gB expression was present in the thymus, spleen, and bursa. At 28 and 35 days PI, gB expression was detected in the thymus and spleen. At 70 days PI, gB expression was detected only in the spleen, and at 105 days PI, gB expression was not detected in any of the lymphoid tissue (thymus, spleen, or bursa). gB expression was not detected in the brachial plexus, sciatic plexus, or feather tips at any of the five time points. The bursa contained HVT only at 21 and 28 days PI. However, HVT was demonstrated in all other tissues from 21 to 105 days PI. Progression from a productive HVT infection to a latent HVT infection results in the loss of gB expression. Throughout this progression, a region of the HVT genome can be detected by appropriate methods.

Octreotide decreases connective tissue formation and improves vascular changes associated with hepatic schistosomiasis.

In clinical practice, Octreotide has its greatest impact in the management of bleeding varices. The present work is the first one which was undertaken to investigate the possible use of Octreotide as an antifibrotic agent and to study its effect on hepatic vasculature in Schistosoma mansoni infection. The material of this investigation consisted of two groups of albino mice (A&B) subdivided each, into normal control, infected control, Octreotide treated, Praziquantel treated and Octreotide with Praziquantel treated subgroups. Groups A & B were sacrificed at the 8th week and the 18th week post infection respectively. By analysis of the obtained results, Octreotide has induced reduction of the portal pressure, the weight of the spleen and the liver, the number of liver egg load, granuloma size and cellularity, and of the degree of hepatic fibrosis quantified by serum PIIINP, serum laminin and tissue collagen using sirius red dye assay. Moreover, the biochemical state of hepatocytes has been improved. The subgroups treated with Octreotide in association with Praziquantel revealed better results than the subgroups treated with Praziquantel alone. Data were analysed in terms of histological extent of liver fibrosis in sections stained with Masson trichrome and sirius red, hepatocytic and sinusoidal changes at an ultrastuctural level and by immunohistochemical demarcation of endothelial cells of blood vessels through the determination of factor VIII related antigen. The promising results detected in this study may encourage to further investigate the positive findings of this drug with the intention of its possible application on a clinical level.

Analysis of acute mechanical insult in an animal model of post-traumatic osteoarthrosis.

Chronic degeneration of articular cartilage and bone in a rabbit model of post-traumatic osteoarthrosis has been hypothesized to occur due to acute stresses that exceed a threshold for injury. In this study, we impacted the rabbit patellofemoral joint at low and high intensities. High-intensity impacts produced degenerative changes in the joint, such as softening of retropatellar cartilage, as measured by indentation, an increase in histopathology of the cartilage, and an increase in thickness of subchondral bone underlying the cartilage. Low-intensity impacts did not cause these progressive changes. These data suggest that low-intensity impacts produced acute tissue stresses below the injury threshold, while high-intensity impacts produced stresses that exceeded the threshold for disease pathogenesis. This study begins to identify "safe" and "unsafe" ranges of acute tissue stress, using the rabbit patella, which may have future utility in the design of injury prevention devices for the human.

Dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior.

The pathogenesis of filariasis has generally been attributed to either physical presence of the adult parasites or the host's immune response to the parasites. However, the spectrum of filariasis cannot be entirely explained by these causes, and other mechanisms must be operative. It is now evident that factors released by filarial parasites likely contribute to the pathogenesis of filarial diseases. Adult heartworms (Dirofilaria immitis) reside in the right heart and pulmonary artery, so the pulmonary artery should be exposed to the highest concentration of filarial factors. We tested the hypothesis that endothelium-dependent relaxation is altered in the in vitro pulmonary artery from heartworm-infected dogs. Relaxation responses to endothelium-dependent vasodilators (methacholine, bradykinin, substance P, and A-23187) and the nonendothelium-dependent vasodilator nitroglycerin and contractile responses were measured in rings of pulmonary artery from control and heartworm-infected dogs. Endothelium-dependent relaxation was assessed in the presence and absence of inhibitors of nitric oxide synthase, cyclooxygenase, and guanylate cyclase. Responses to methacholine, substance P, and A-23187, but not to bradykinin, nitroglycerin, norepinephrine, or KCl, were depressed in pulmonary artery from heartworm-infected dogs when compared with control, suggesting that changes in endothelial cell and not vascular smooth muscle behavior are involved in altered relaxation. The mechanism of endothelium-dependent relaxation in control pulmonary artery appears to involve nitric oxide in the case of methacholine and both nitric oxide and a cyclooxygenase product in the case of bradykinin and A-23187. The mechanism of endothelium-dependent relaxation in pulmonary artery from heartworm-infected dogs was not clearly elucidated. These data provide no evidence that heartworm infection globally influences either endothelial cell receptor function or the vascular smooth muscle guanylate cyclase guanosine 3',5'-cyclic monophosphate system, making it likely that changes in intracellular signaling are primarily responsible for the observed alteration of endothelium-mediated relaxation. Alteration of endothelial cell function by filarial parasites may be an important component in the pathology associated with filariasis.

A comparative study of histological conditions suitable for both immunofluorescence and in situ hybridization in the detection of Herpesvirus and its antigens in chicken tissues.

Our objective was to identify an optimal single set of conditions for use in both indirect immunofluorescence assays (IFA) and in situ hybridization (ISH) to detect viral proteins and nucleic acids in avian lymphoid and neural tissues. Various fixatives were evaluated for use with IFA to detect turkey Herpesvirus (HVT) glycoprotein B (gB) and ISH to identify HVT mRNA in chicken tissues. A precipitating fixative (acetone) was compared to crosslinking fixatives [buffered glutaraldehyde-picric acid (BGPA), 10% formalin, and 4% paraformaldehyde] for both IFA and ISH using spleen, thymus, bursa, sciatic plexus, and brachial plexus of 28-day-old chickens. Four percent paraformaldehyde was found to be the optimal fixative for preservation of all chicken tissues examined with both IFA and ISH. Glass slide preparation, incubation temperatures, and tissue processing were each individually evaluated for ISH and IFA. Silylated slides provided the best retention of tissue sections for both procedures. For IFA, 37 degrees C was the ideal incubation temperature tested, whereas the optimal incubation temperature tested for ISH was 47 degrees C. Of the blocking agents compared, Evans blue dye prevented background fluorescence to a greater extent than either calf serum or bovine serum albumin. These findings provide a technical basis for investigations into various aspects of the molecular pathology of avian diseases.

Leukotriene C(4) biosynthesis in isolated August rat peritoneal leukocytes.

The mixed leukocyte population obtained from the peritoneum of the August rat is a potentially important experimental model of inherent eosinophilia that has not been well characterized. In the present study, isolated cell preparations generated a concentration-dependent release of leukotriene (LT) C(4) when exposed to the Ca(2+) ionophore A23187, reaching maximal stimulation at 5.0 muM. This response was inhibited by the 5-lipoxygenase activating protein antagonist MK-886 (0.1 muM), nominally Ca(2+) and Mg(2+)-free incubation media and by activation of protein kinase C via phorbol 12-myristate 13-acetate (50 nM). These findings establish a model system for investigating LTC(4) profiles contingent with innate peritoneal eosinophilia and are consistent with the hypothesis that cellular LTC(4) biosynthesis is phosphoregulated.

Ivermectin treatment in severe asymmetric reactive onchodermatitis (sowda) in Sudan.

Ivermectin efficacy and post-treatment reactions in asymmetric severe reactive ochodermatitis (sowda) were studied in 8 patients with sowda syndrome and 6 with mild generalized onchodermatitis in Sudan. Initial skin snips from 12 patients contained microfilariae (1-9 per mg skin). Patients were treated in hospital with a single oral dose of c. 150 micrograms/kg ivermectin (103-200 micrograms/kg) and monitored for frequency and severity of post-treatment reactions for 4 weeks. Serial samples of heparinized blood were collected over the first 24 h after treatment for determination of ivermectin pharmacokinetics. Skin snips from all patients on days 3 and 28 revealed no microfilariae. Post-treatment reactions were more common and severe in individuals with sowda; they consisted mainly of musculoskeletal pain, local swellings with pitting oedema, and lymph gland tenderness and enlargement. No relation was established between these reactions, the microfilarial infection intensity, or the plasma pharmacokinetic profiles. A single oral dose of ivermectin cleared the skin of microfilariae and led to improvement of symptoms and dermatological signs of sowda, but resulted in more marked reactions than in cases of generalized onchodermatitis.