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A method is described to isolate microfilariae of Litomosoides carinii on a Ficoll isopycnic gradient. The simple and fast procedure yields a high percentage of microfilariae apparently without causing any alteration of the larval surface and vitality.
After intracutaneous inoculation of BCG and challenge by subcutaneous injection of infective larvae of Litomosoides carinii, the parasitaemia of the filarial infection in cotton rats remains significantly lower when BCG and larvae are applied in the region of the same popliteal and ileal lymph nodes. However, when the infective larvae are directed to other regional lymph nodes (Ln cubitales and axillares), the depression of microfilaraemia is missed. The worm load (recovery rate) and the expulsion of microfilariae by the adult worms are not influenced by the BCG inoculation. Obviously BCG stimulates the lymphatic tissue unspecifically, and the infective larvae produce the first antigen contact, which is boostered by the microfilariae at the onset of patency. When the intracutaneous BCG inoculation is combined with specific antigen stimulation by simultaneous injection of blood microfilariae in the region of the same lymph nodes, the microfilaraemia of the challenge infection disappears completely or remains extremely low.
Crude aqueous Litomosoides carinii adult worm extract was used as antigen for the detection of antibodies in sera from African patients with proven onchocerciasis (n = 45) resident in rural endemic areas of Togo and Sierra Leone. In 71% of cases this extract was found to produce 1 to 5 precipitation arcs in immunoelectrophoresis. Using a crude aqueous extract from adult Onchocerca volvulus, precipitation tests were positive in 75% of cases. The complexity of the L. carinii crude extract was shown by PAG-disc electrophoresis, PAG-electrofocusing, immunoelectrophoresis and crossed immunoelectrophoresis with the appropriate rabbit-antiserum. An antigen detecting onchocercal antibodies was isolated by two step preparative flat bed electrofocusing in granulated gel (PEGG). The antigen (pI 6.55, molecular weight 55 to 60 kd as estimated by SDS-PAG electrophoresis) was very suitable for antibody demonstration in double diffusion test and immunoelectrophoresis. Preliminary controls for specificity were performed by diffusing the antigen against sera from human and animal helminthoses including filarial infections. In contrast to the crude L. carinii extract no reaction was observed with sera from helminthic infections others than filariasis.
Treatment of Litomosoides carinii infected Mastomys natalensis with diethylcarbamazine (DEC: 500 mg/kg p.o.) was followed by increased occurrence of microfilariae in the bronchi of the host after 40 min and lasting at least until 6 h after treatment. After 4 h, increased levels of larvae were observed in the gut. Only a few microfilariae occurred in the bladder and sputum. Accumulations of microfilariae were found furthermore in the Lnn. hepaticae whereas no changes were observed in the inguinal or jejunal and lung and pleura associated lymph nodes. Increased numbers of microfilariae were found in the peritoneal cavity only after 8 and continuing until at least 48 h after treatment. In contrast, after haloxon treatment (100 mg/kg p.o.) an accumulation of microfilariae was found in the peritoneal cavity only, following a time course similar to that after DEC.
Antibodies inhibitory to the activity of the enzyme phosphoglucomutase (PGM) EC 2.7.5.1. of Brugia pahangi adult worm were demonstrated in sera from five rabbits immunized against this filarioid and from 5 of 27 cats infected with B. pahangi, by isoelectricfocusing and spectrophotometric techniques. This anti-PGM activity was species- and stage-specific. This raises the possibility of using species-specific isoenzymes of parasitic origin as antigens in serological tests.
The significance of the histological changes induced by filarial larvae in the indirect flight muscle fibres of susceptible female mosquitoes was investigated by inflicting mechanical (needle-puncture) injury on thoracic tissues of male and female mosquitoes of various species. Mechanical injury caused the flight muscle of all mosquitoes, irrespective of species, sex or filarial susceptibility, to react in the same way as that of filaria-infected, susceptible females; other thoracic tissues responded in a similar fashion. Since these responses are closely akin to those observed by earlier authors in various tissues of other insects subjected to experimental trauma, it seems reasonable to regard the reactions of indirect flight muscle of mosquitoes to filarial larvae as an example of a common, non-specific response by insects to a range of traumatic stimuli.
Effects of piperazine derivatives, especially of diethylcarbamazine (DEC) on adult Angiostrongylus cantonensis and Dirofilaria immitis were examined. Piperazine (3 X 10(-5)-10(-4) M) paralyzed A. cantonensis and the action was antagonized by picrotoxin. 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (10(-5)-10(-4) M) caused contraction but little effect was produced by strychnine. An inhibitory effect on untreated preparations was caused by lower concentrations (3 X 10(-6)-10(-5) M) of diethylcarbamazine citrate (DEC) and also on the preparations contracted by eserine. A stimulatory effect was also seen when higher concentrations (10(-4)-3 X 10(-4) M) of this drug were applied to both preparations. The inhibitory action of DEC was antagonized by gabergic antagonists such as picrotoxin and bicuculline, but not by alpha-adrenergic antagonists like dibenamine and phentolamine. When the worm preparation was paralyzed by strychnine or hexylresorcinol (inhibitors of the release of acetylcholine in this worm), the stimulatory effect of DEC was blocked, but pyrantel (a nicotinic cholinergic agonist) contracted the paralyzed preparation. However, the effect of DEC on D. immitis (10(-7)-3 X 10(-4) M) was inhibitory, and this action was also antagonized by picrotoxin. These results suggest that the DEC inhibitory and stimulatory action is through the gabergic and cholinergic mechanisms in adult A. cantonensis and D. immitis.
The efficacy of ivermectin (Iv) was evaluated against four species of filariae, Litomosoides carinii, Acanthocheilonema viteae, Brugia pahangi and Brugia malayi in Mastomys natalensis. Animals with patent infections, induced with L3 larvae, by intravenous (iv) infusion of the respective microfilariae (Mf) (5 x 10(4) Mf per animal) or by intraperitoneal (ip) route (2 x 10(4) Mf per animal) were used in this study. A single dose of Iv (100 micrograms.kg-1) given subcutaneously (sc) to Mastomys infected with L. carinii or A. viteae resulted in the disappearance of microfilaremia within 2 h of treatment. Iv treatment of sc-infected animals with Brugia spp. had no immediate effect on the circulating Mf 60 days post-treatment. In contrast, such treatment of animals infected with Mf by intravenous infusion completely eliminated the larvae of all four species from the circulation. Iv treatment had no significant effect on the Mf of L. carinii, B. pahangi and B. malayi in animals infected by the ip route. However, the drug had dramatic effect in killing the Mf of A. viteae in the peritoneal cavity. Sera from Iv-treated normal or from L. carinii- or A. viteae-infected Mastomys were effective in clearing the circulating Mf of the species when administered to animals with the respective infections. Similar rapid clearance of Mf was seen when the sera were administered to animals infected iv with these larvae. Furthermore, adult females of L. carinii and A. viteae recovered from Mastomys on different days after Iv treatment released smaller numbers of Mf in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
The antigenic cross-reactivity of the excretions-secretions (E-S) of Litomosoides carinii was investigated. Immunoprecipitation using pooled sera from a number of human filarial infections in combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed variations in the cross-reactivity of individual molecules. Some were specific to L. carinii, the major examples in this category being two E-S components of 140 and 160 kDa released by day 40- to 42-day-old female worms. Another, a high-molecular-weight product of 26- to 28-day E-S, was broadly cross-reactive. A third group appeared to exhibit reactivity to antibody to some but not all human filarial parasites. The most striking examples of this were two distinct 14-kDa products that bound solely to antibodies in an onchocerciasis serum pool. These results are discussed in relation to the use of cross-reacting molecules in investigating the immunisation potential, defining the function, and evaluating the diagnostic utility of human filarial E-S.
Litomosoides carinii-, Acanthocheilonema viteae- or Brugia malayi-infected Mastomys natalensis were sensitised against sheep red blood cells (SRBC) on various occasions after infection to determine the effect of filarial infections on the immune response to a non-filarial antigen. The phagocytic activity of the reticuloendothelial system (RES) was controlled in vivo by the elimination of 51Cr-labelled SRBC. Antibody titres against SRBC (agglutinating and lytic antibodies) were similar to those of uninfected controls in L. carinii- or B. malayi-infected Mastomys sensitised during prepatency or early patency up to 90 days post infection (p.i.) but were reduced in animals sensitised during patency. A significant inverse correlation existed between anti-SRBC antibody titres and microfilaraemia levels. In contrast, A. viteae-infected Mastomys showed reduced humoral anti-SRBC responses at the end of prepatency, whereas the response tended towards normal with increasing parasitaemia. Delayed-type hypersensitivity (DTH) against SRBC was measured as footpad swelling after sensitisation by the s.c. or i.v. route and intraplantar challenge. DTH reactions were reduced during prepatency in all infections after s.c. sensitisation. During patency, 24-h reactions were similar to those of age-matched controls but the swelling persisted 24 or 48 h longer than in the latter. In A. viteae infections, even enhanced 24-h reactions were found during patency. Histological investigations did not reveal differences in the type of cell infiltrations between infected and control animals. After i.v. sensitisation with SRBC, L. carinii- and A. viteae-infected animals showed weaker DTH reactions than the controls, independent of the period after infection. In the case of B. malayi infections, DTH reactions were similar to those of controls during early prepatency, whereas reduced DTH responses were observed later than 50 days p.i. As shown in L. carinii-infected animals, depressed DTH reactions after i.v. sensitisation did not depend on an altered expression phase but rather on an altered regulation during the inductive phase of the response: increases in the sensitising SRBC doses that caused decreasing DTH reactions in uninfected animals led to enhanced reactions in infected animals. Phagocytosis of i.v. injected 51Cr-labelled SRBC was enhanced during prepatency in L. carinii infection and during patency in all infections.
When microfilariae (Mf) of Dirofilaria immitis, both uterine and systemic, were incubated in an in vitro cytotoxicity assay with neutrophils and sera from dogs with occult infections, some Mf remained free of adherent cells and consequently evaded cytotoxicity. The ability to evade cytotoxicity could not be related to the age of the Mf, and host albumin was not detected on any Mf, either uterine or systemic. However, it was shown that some Mf failed to bind IgM, IgG and C3 when incubated with occult sera. It is suggested that the ability of some Mf to evade serum-dependent, neutrophil-mediated cytotoxicity in vitro was related to differences in their antigenicity.
Electron microscopy coupled with ferritin-conjugated indirect immunolabeling was used to locate sites of adsorbed host protein on cuticular surfaces of the adult canine heartworm, Dirofilaria immitis. The epicuticle appeared as a trilaminated structure. At high magnifications, the outermost layer of this structure was resolved into a trilaminar layer, which might correspond to the plasma membrane of animal cells. A ruthenium red-positive layer was external to the epicuticle. Ferritin-antibody conjugates showed evidence of adsorbed dog albumin, dog immunoglobulin class G (IgG), and dog complement fraction 3 (C3) on the surface. Ferritin adsorption to control surfaces was minimal. Possible causes and effects of interfacial host-protein adsorption are discussed in an attempt to bring insight to the hemocompatible nature of the parasitic cuticle.
Patterns of codon usage and bias were compared among taxa of the genera Brugia, Echinococcus, Onchocerca and Schistosoma by metric multidimensional scaling and three commonly used indices of bias: Nc, GC3S and B. The overall codon usage for each taxon was compared, as was the codon usage for each individual gene within the taxa. Differences in the patterns of codon usage observed between taxa were dependent on the overall base composition of the genes analysed. The codon usage of Echinococcus was distinct from that of the other taxa. Furthermore, the pattern of codon usage detected by the average codon usage summed across all genes for each taxon was not shown by all genes from that taxon.
Microfilarial sheaths of Litomosoides carinii were isolated and extracted with 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol (2ME). Extraction with SDS alone did not alter the ultrastructure of the sheaths and yielded five polypeptides (27-67 kDa) that were not recognized by antibodies of infected hosts but reacted with antibodies to host-serum proteins. 2ME treatment caused partial solubilization of the sheaths (45% as determined by amino acid analysis), which could be further improved by combining 2ME with SDS. The remainder showed filamentous/threadlike structures on electron microscopic examination. As compared with whole sheaths, the insoluble proportion was markedly enriched in alanine and cysteine but contained less galactosamine, serine, and threonine. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 2ME/SDS-extractable components showed 12-16 bands of 14- greater than 120 kDa. A predominant component had an apparent molecular mass of 22 kDa. Two bands (42 and 120 kDa) could be stained with Coomassie blue but showed "negative" staining when gels were stained with silver. Several components (but not the 22-kDa polypeptide) bore phosphocholine epitopes. Apart from the negatively staining bands, most of the 2ME-soluble sheath components were recognized by antibodies of L. carinii-infected Mastomys coucha. Except for several polypeptides that had been unspecifically recognized by IgM, the antibody response to sheath components started at the end of the prepatent period.
Three cytotypes of Foleyella agamae were isolated from naturally infected wild-caught Agama agama in Nigeria. Cytotype A (2n = 8) has a body length/oesophageal length ratio (L/O) of 87.6 +/- 14.2 in female worms and a large somatic size (length, 63.8 +/- 12.7 mm in female worms and 25.5 +/- 3.4 mm in males). Cytotype B (2n = 6) has an L/O of 43.4 +/- 6.6 in females and a small to average somatic size (length, 36.7 +/- 11.9 mm in females and 19.5 +/- 2.3 mm in males). Cytotype C (2n = 4) has an L/O of 23.8 +/- 8.2 in female worms and a small somatic size (length, 18.8 +/- 3.8 mm in females and 14.1 +/- 3.0 mm in males). No cytotype with odd numbers of chromosomes was observed. Comparisons with similar types of speciation in medically important filarial parasites were drawn. Introgressive hydridisation in the speciation of filarial parasites is postulated.
Spermine and spermidine were found to be the principal polyamines in the bovine filarial parasite Setaria cervi, whereas putrescine was observed in very low amounts. Studies conducted on the enzymes of polyamine biosynthesis revealed low activity for S-adenosyl-methionine decarboxylase, questionable and negligible activities for the decarboxylation of ornithine and arginine, and appreciable activity for ornithine aminotransferase. Uptake studies with radiolabeled putrescine, spermidine and spermine showed that these amines are rapidly taken up from the medium by an active uptake process. The uptake was temperature-sensitive and abolished at 0-4 degrees C. The questionable presence of biosynthetic enzymes such as ornithine and arginine decarboxylase and, on the other hand, an effective uptake mechanism indicate that the parasite may depend on the host for its polyamine requirement, thereby indicating a possible target for chemotherapy.
Alterations in the fine structures of female Brugia spp. and Litomosoides carinii were investigated after in vivo treatment with curative doses of 4 compounds: CGP 20376 [2-tert-butyl-benzothiazole-5-methoxy-6-dithiocarbamic-S-(2- carboxyethyl)-ester], CGP 21833 (2-tert-butyl-benzothiazole-5-methyl-6- N-methylamino-piperazinylthiocarbonylamide), CGP 6140 [4-Nitro-4'-(N-methyl-piperazinylthiocarbonylamido)-diphenylamine] and amoscanate (4-isothiocyanato-4'-nitrodiphenylamine). All compounds caused early alterations in the somatic muscle cells. These alterations usually appeared within 24 h after treatment; they occurred later only after treatment of L. carinii with amoscanate. In Brugia spp., swelling of the muscle cells occurred in which the glycogen deposits considerably increased in size. The electron density of the cytoplasm surrounding the myofilaments in the fibrillar portion of the muscle cells increased, and light zones appeared between the fibrils. The muscle cell mitochondria swelled, particularly their inner matrix, which became more electron-lucent, with some dense spots. In L. carinii the muscle cells were not increased in size, but their mitochondria were considerably swollen before disintegration; this was followed by disintegration of the myofilaments and vacuolization of the cytoplasm. Vacuolization before mitochondrial swelling was observed only after treatment with CGP 6140. Other tissues of this species were not altered before the 2nd day after treatment. In Brugia spp., electron-lucent appeared in the hypodermis either simultaneously with the alterations in the muscle cells or a few hours later. At 24 h after treatment with amoscanate, blebs were formed on the luminal side of the intestinal membrane.
Female filariae of the species Brugia malayi and Litomosoides carinii were investigated by means of electron microscopy after in vivo treatment with flubendazole. The earliest fine-structure alteration in both species was the disappearance of microtubuli from the intestinal cells as soon as 6 h after treatment. There was no further disintegration of intestinal cells for several days. Microtubuli disappeared from the outer zone of the hypodermal cytoplasm 24 h after treatment. At this time, marked alterations were also observed in the oogonia and in the embryonic cells. Many of these were swollen; their nuclear envelope was partly resolved and the chromatin was condensed, but no spindle apparatus was formed. The early fine-structure alterations observed after in vivo treatment with flubendazole consisted of the disappearance of microtubuli from various tissues. This led to the interruption of cell division in oogonia and embryonic cells and, and subsequently, to the disintegration of most other filarial tissues. These morphological alterations differed considerably from those observed after treatment with benzothiazole derivatives, which do not affect the microtubuli of the filariae.