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Histopathological studies have provided circumstantial evidence that helminth parasite destruction occurs in the lung; however controlled in vitro studies on the helminthocidal activity of lung cells have not been reported. This study presents evidence that Nippostrongylus brasiliensis infection in the rat induces alterations in broncho-alveolar lavage (BAL) cell numbers, differential counts, and in vitro helminthocidal activity. Normal, uninfected rats yielded 33 ± 0.6 × 106 BAL cells/rat, consisting predominantly of alveolar macrophages (>90%). However on days 2-8 post-infection there was a 1.5-2.4-fold increase in BAL cell numbers with a significant neutrophilia on day 2 and a significant increase in the absolute number of all cell types on day 8. On day 32 post-infection, BAL cell numbers had returned to control levels. Normal BAL cells neither adhered to nor killed N. brasiliensis infective larvae (L3) in the presence of rat complement. By contrast BAL cells recovered from infected rats on days, 2, 8 or 32 post-infection (D2, D8 and D32 BAL cells, respectively) adhered under similar conditions. However, only D8 and D32 BAL cells killed L3. This complement-dependent killing correlated with significantly increased numbers of C3 receptor bearing alveolar macrophages in D8 and D32 BAL cells. Complement-dependent alveolar macrophage helminthocidal activity may therefore play an important role in lung resistance against resident or migrating helminths. [ABSTRACT FROM AUTHOR]

Alveolar macrophages (AM) of rats infected with 3000 Nippostrongylus brasiliensis infective larvae for 2, 8 or 32 days (D2, D8 or D32 AM) quantitatively surpassed AM from uninfected rats in one or more of IgG- or C3-dependent phagocytosis indices, β-D-glucuronidase release, or spontaneous release of thymocyte activating factor (interleukin-1, IL-1) and hepatocyte stimulation factor (HSF), These observations suggest that N. brasiliensis infection results in the activation of AM. We have reported previously that a greater proportion of AM from infected rats expressed C3 receptors and were helminthocidal in vitro in the presence of complement than normal AM which were not helminthocidal. The acquisition of the activated state by AM during infection may play a role in in vivo lung resistance against migrating helminth parasites. [ABSTRACT FROM AUTHOR]

The vertebrate host, constantly threatened and invaded by various pathogens, has evolved two mechanisms of resistance. The first and phylogenetically oldest mechanism is the non-specific protection provided by complement alone or in concert with phagocytes such as neutrophils and macrophages. The second and evolutionarily later mechanism is the specific immune response involving antibodies, lymphocytes and macrophages. Both mechanisms serve the same function — to protect the host against pathogens.

The major objective of this study was to evaluate the usefulness of IgG4 ELISA and Western blot analysis, using a crude extract of Onchocerca volvulus adult worms as antigens, for diagnosing onchocerciasis in a Gabonese paediatric population with mixed filarial infections. The subjects had loaisis, streptocercosis or mansonellosis in addition to onchocerciasis. Control sera from loaisis or mansonellosis subjects residing outside the endemic zone were used to provide the cut-off point for positive results. The IgG4 ELISA had a specificity of 96% but a lower sensitivity of 78.7%. It detected 25 onchocerciasis eases out of 65 individuals who were negative on parasitological examination. Furthermore, the ELISA provided a more accurate picture of onchocerciasis transmission in a village with very low skin microfilarial load. A 27.5-kD antigen was identified on Western blots as a marker of onchocerciasis. The paediatric population provided a reliable window for assessing the parasitologic and serologic parameters in the three villages with disparate levels of onchocerciasis transmission. [ABSTRACT FROM AUTHOR]

The proliferation and cytokine profiles of peripheral blood mononuclear cells (PBMC) from microfilaraemic (Mf+) subjects infected by Loa loa in response to antigens of several parasitic stages were compared with those from amicrofilaraemic (Mf+) individuals. While a strong lymphoproliferative response and consistent levels of both Th1 (IL-2, interferon-gamma (IFN-γ)) and Th2 (IL-4, IL-5) type cytokines were observed in response to adult worm (AW) and microfilariae (Mf) antigen in Mf- individuals, Mf+ subjects were characterized by a T cell unresponsiveness. including proliferation. cytokine production and IL-2 mRNA expression. Conversely, T cell responsiveness to mitogens and non-specific antigen were similar in the two endemic populations. Depletion of lymphocyte subpopulations indicated that T CD4+ were mainly involved in the specific cellular response. In contrast to other cytokines, IL-10 was produced in response to all parasitic stages, in both Mf+ and Mf- patients. Neutralization of IL-10 did not restore cytokine production in Mf+ patients, while B7 mRNA expression was similar between Mf+ and Mf- subjects in response to Mf antigen, suggesting that IL-10 was not the only factor responsible for T cell unresponsiveness. Mf+ patients have lower Mf antigen-specific IgG levels compared with Mf- and there is a significant correlation between Mf antigen-specific antibodies and IL-5 responses. These findings suggest that Mf- status is correlated with T helper responsiveness, including proliferation and production of both Th1- and Th2-type cytokines, whereas Mf+ status is characterized by unresponsiveness of the same cell population, induced and or maintained by microfilariae. [ABSTRACT FROM AUTHOR]

Human filariasis due to Loa loa differs from other filariasis in that the majority of infected subjects are without circulating microfilariae (occult loiasis). In search for alternative diagnostic methods, which do not depend on circulating microfilariae or the (rather infrequent) eye-passage of adult worms, it was shown earlier that IgG4 antibodies directed against Loa loa adult worm antigen are apparently a good marker of occult loiasis and specific with regard to the sympatrically occurring Mansonella perstans. In this study we evaluated an IgG4 antibody-based ELISA using crude extract of Loa loa microfilariae (which is easier to obtain than adult worm) to estimate the prevalence of loiasis in 3 villages in South-East Gabon. Of 222 examined individuals (80 children < 16 years, 142 adults) 44 (20%) carried Loa loa microfilariae and 170 (77%) M. perstans. Using the mean OD-value + 1 standard deviation of 9 sera from patients solely infected with M. perstans (from the Gambia, where Loa loa is not endemic) as a cut-off, 35 of the 44 microfilaraemic Loa loa patients and 2 of the 9 Gambian controls were positive. This shows that our method had a sensitivity of 80% and a specificity of 78%. Among the remaining 178 subjects who had no microfilariae of Loa loa, as many as 97 (55%) had significant levels of specific IgG4 antibodies against Loa loa, suggesting that they carried occult loiasis. The mean IgG4 level in these putatively occult loiasis patients was slightly but significantly lower than in microfilaraemic subjects (P < 0.03). In conclusion, despite the limited sensitivity and specificity of our method, IgG4- ELISA at present is a very useful tool in estimating the real prevalence of loiasis in epidemiological surveys and at the individual level can confirm the diagnosis of L. loa amicrofilaraemic subjects with clinical signs suggesting loiasis. [ABSTRACT FROM AUTHOR]

A cross-sectional study of pregnant women was conducted at Nsambya Hospital in Kampala, to investigate the prevalence and effect of Plasmodium falciparum infections during pregnancy, in a peri-urban/urban location. Overall, 544 pregnant women were recruited when they presented at the labour ward for delivery. After giving informed consent, each subject answered a questionnaire and underwent a physical examination, and peripheral-blood samples were obtained. After each uncomplicated delivery, samples of placental and cord blood were obtained from the placenta and infant, respectively, and infant birthweights were recorded. Smears were prepared from the blood samples and checked for parasites. Only 46 and 36 of the 537 women investigated were positive for P. falciparum infection in their peripheral and placental blood, respectively. Plasmodium falciparum was the only parasite encountered. The prevalences of low birthweight and maternal parasitaemia and the intensities of maternal infection were each greater in primigravidae than secundi- or multi-gravidae. Despite the low prevalence of parasitaemia in this population, P. falciparum infection in the primigravidae was a significant contributor to their ill health, leading to low birthweights in their infants. [ABSTRACT FROM AUTHOR]

Mutations in the dihydrofolate reductase gene (dhfr) of Plasmodium falciparum have been proposed as molecular markers for the surveillance of sulfadoxine-pyrimethamine (SP)-resistant malaria, but such proposals have not been validated. At 7 Ugandan sites in 1999, we determined the population-based prevalence of infections with mutations and the mutant allele frequency of dhfr codons 108, 51, and 59 using a random sample of infected individuals aged 1-45 years. Sulfadoxine-pyrimethamine treatment failure was independently estimated by in vivo tests in 327 children aged 6-59 months with clinical malaria. The prevalence of infections with the single point mutations and the dhfr codons 108 and 51 mutant allele frequency were not correlated to SP treatment failure. However, the dhfr codon 59 mutant allele frequency was positively correlated to SP treatment failure (r = 0.72, P = 0.06). The ratio of the infections with the mutant to wild genotype (M/W) and that of the mutant to wild allele (MA/WA) had the same values. Both dhfr codon 59 M/W and MA/WA ratio were significantly and positively correlated to SP treatment failure (r = 0.73, P = 0.05). Moreover, the prevalence of infections with only 2 mutations (Asn-108 plus Ile-51) was significantly and inversely correlated to the prevalence of infections with 3 mutations (Asn-108 plus Ile-51 plus Arg-59) (r = 0.92, P = 0.004), suggesting the stepwise accumulation of the dhfr mutations is Asn-108 Ile-51 Arg-59 and further supporting the idea of using the dhfr codon 59 M/W ratio as a molecular index for the prediction of SP treatment failure. Atthe population level, the dhfr codon 59 M/W ratio is a simple and stable index for the estimation of SP treatment failure.

Recent molecular studies of chloroquine (CQ) resistance of Plasmodium falciparum have demonstrated an association between a mutation in the PfCRT gene and CQ resistance. We identified wild type and mutant alleles of the PfCRT codon 76 in baseline pre-CQ treatment P. falciparum isolates collected during 1999 and investigated their relationship to CQ efficacy in 3 different sites with different levels of CQ parasite resistance in Uganda. Of 32 isolates from Mulago Hospital, all were mutant (100%), while of 45 isolates from Tororo, 5 (11%) were mixed wild type and mutant and 40 (89%) were mutants only. Of 41 isolates from Apac, 13 (32%) were mixed wild type and mutant whereas 28 (68%) were mutants only. The finding of 100% prevalence of the Thr-76 mutant allele in all isolates at the 3 sites was remarkable. We found no association between the presence of Thr-76 mutation and treatment outcome at all the sites. However, the prevalence of the wild-type Lys-76 allele was higher in Apac, an area with lower CQ parasite resistance, compared to Tororo and Mulago which have relatively higher CQ parasite resistance. The Thr-76 allele as a marker of CQ resistance is probably useful in regions where the allele frequency has not yet plateaued.

Objective: To establish Plasmodium falciparum malariometric indices in a field study site in Apac district, northern Uganda., Design: A community-based cross sectional survey., Settings: Atopi Parish, Apac district, Uganda, 1995., Subjects: One thousand two hundred and thirty four volunteers aged below one and ninety years., Main Outcome Measures: P. falciparum parasitaemia rates and parasite density, splenomegaly, bednet use and chloroquine consumption., Interventions: All subjects with P. falciparum positive smears were treated with chloroquine., Results: The population prevalence of parasitaemia was 62.1% with the predominant species being P. falciparum (100%) and P. malariae in the minority (3.5%); P. ovale was not seen. The prevalence of parasitaemia in subjects older than 20 years and in those under ten years was 36% and 85%, respectively. The geometric mean parasite density started to decline by the age of six years. The splenomegaly rate in subjects over the age of 12 years and in those under nine years was 19.8% and 63.1%, respectively. Bednet use and chloroquine consumption was low. Interestingly, the reported use of chloroquine in the week immediately preceding the study was more frequent in children under two years old than in the rest of the population., Conclusion: Malaria transmission in Atopi Parish in northern Uganda is hyperendemic and age-related acquired anti-parasite immunity seems to appear by seven years of age.

Objective: To determine the natural human humoral immune responses to the 19 kilodalton carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)), a malaria candidate vaccine antigen and to determine the prevalence of MAD20 and K1 alleles of P. falciparum MSP1., Design: Community based cross-sectional study., Setting: Atopi Parish, Apac District, Uganda, 1995., Subjects: Three hundred and seventy four Ugandans between <1 and 70 years old provided serum samples., Main Outcome Measures: IgG subclass antibodies by ELISA; MAD20 and K1 allelic types of MSP1 by PCR., Results: Both the prevalence and the mean concentration of serum IgG1, and to a lesser extent IgG3, antibodies increased with age. IgG2 or IgG4 antibodies were virtually nonexistent. The cross-reactivity between the 4 sequence variants (E-KNG, E-TSR, Q-KNG and Q-TSR) of MSP1(19) was confirmed; however, a minority of sera preferentially recognised the KNG but not the TSR variants. All 33 P. falciparum isolates from different parts of Uganda carried the E-TSR (Mad20) allelic type and 3 isolates were mixed infections with E-TSR (MAD20) and Q-KNG (K1) allelic types, confirming the rarity of the K1 allele in Uganda., Conclusion: There is a robust IgG1 antibody response to the malaria vaccine candidate antigen MSP1(19) which begins at an early age. Future cohort studies are necessary to estblish the impact of these antibodies on clinical immunity to malaria. The MAD20 allelic type of MSP1 id predominant in Ugandan P. falciparum isolates.

A cross-sectional study of pregnant women was conducted at Nsambya Hospital in Kampala, to investigate the prevalence and effect of Plasmodium falciparum infections during pregnancy, in a peri-urban/urban location. Overall, 544 pregnant women were recruited when they presented at the labour ward for delivery. After giving informed consent, each subject answered a questionnaire and underwent a physical examination, and peripheral-blood samples were obtained. After each uncomplicated delivery, samples of placental and cord blood were obtained from the placenta and infant, respectively, and infant birthweights were recorded. Smears were prepared from the blood samples and checked for parasites. Only 46 and 36 of the 537 women investigated were positive for P. falciparum infection in their peripheral and placental blood, respectively. Plasmodium falciparum was the only parasite encountered. The prevalences of low birthweight and maternal parasitaemia and the intensities of maternal infection were each greater in primigravidae than secundi- or multi-gravidae. Despite the low prevalence of parasitaemia in this population, P. falciparum infection in the primigravidae was a significant contributor to their ill health, leading to low birthweights in their infants.

By evaluating the diagnostic methods developed in our laboratory, the prevalence of loaiosis was estimated among 201 individuals from the province of Haut Ogooué in Gabon using IgG4 serology and nested-PCR. The study showed that the prevalence of loaiosis was higher than that described using standard microscopy. IgG4-based ELISA (Enzyme Linked Immunosorbant Assay) using crude extract of Loa loa microfilariae showed that 80% (35/44) of microfilaraemic individuals (MF') and 56% (88/157) of amicrofilaraemics (AMF) presented antibodies. By contrast, L. loa specific DNA amplified by nested-PCR was detected in all MF and in 68% (106/157) of AMF. Among the 201 samples tested, 95 (47%) gave positive results in both tests. These results indicate that the presence of IgG4 antibodies directed against crude extract of L. loa microfilariae is not linked to the positivity of nested-PCR assay (chi 2 for paired data = 8.78; P < 0.02). We conclude that the PCR assay is more sensitive than the detection of IgG4 antibodies (directed against crude extract of L. loa microfilariae) in detecting loaiosis, and particularly occult loaiosis (infection without circulating microfilariae).

The human and simian strains of Loa loa microfilariae are morphologically identical even though their periodicities vary. When using primate models (Mandrillus sphinx) of human loaisis for vaccination trials, the absence of any ongoing simian L. loa infection must be demonstrated. Nested primers derived from a human strain of L. loa (targeted on the repeat 3 region of the gene encoding the 15 kDa polyprotein; 15r3) amplified at 366 bp sequence from simian L. loa genomic DNA and blood lysates from mandrills infected with simian L. loa. This nested-PCR assay has been tested on 12 amicrofilaremic (AMF) mandrills (without filarial microfilariae) and was positive in four mandrills. The nested-PCR product derived from simian L. loa genomic DNA and from three of four AMF mandrills has been sequenced. No difference was observed between the four sequences, which, in addition, were 99.18% identical to the 15r3 of human L. loa. Therefore, the 15r3 sequence is conserved within human and simian L. loa. These results suggest that the four PCR-positive mandrills without circulating microfilariae had occult simian L. loa infections. The study demonstrates the ability of a nested-PCR assay to identify animals naturally infected with simian L. loa.

Filarial loiasis differs from other filariases in that most infected subjects are amicrofilaremic. This difference raises the notion of occult infection. The aim of this study was to evaluate the relationship between the intensity of transmission and incidence of infection. For this purpose we determined the incidence of loiasis both microscopically and by PCR in 201 subjects from three villages in the province of Haut Ogooue in Gabon. Intensity of transmission, expressed in ATP (annual transmission potential) in these villages was estimated to be 250 infecting larvae per individual per year (L3/man/yr) in Moyabi, 180 L3/man/yr in N'dokaye, and 43,000 L3/man/yr in Okoumbi. Although there was no significant difference between the three villages with regard to the incidence of microfilaremia (21 p. 100 and 22 p. 100), the incidence of occult infection, i.e., positive PCR in amicrofilaremic subjects, was 45 p. 100 in Moyabi, 79 p. 100 in N'dokaye and 80 p. 100 in Okoumbi. The overall incidence of loiasis was 57 p. 100 in Moyabi and 85 p. 100 in both N'dokaye and Okoumbi. These findings demonstrate that the incidence of loiasis is correlated with the intensity of transmission (p < 0.001), especially in children. Taking this information into account will improve control of Loa loa in endemic areas.

A nested polymerase chain reaction (nested PCR) assay, targeted on the repeat 3 region (15r3) of the gene coding for a Loa loa 15 kD polyprotein, was developed to detect L. loa infection. The assay has a sensitivity of 95% and is 100% specific with regard to sympatric filarial parasites: Mansonella perstans, Onchocerca volvulus and Wuchereria bancrofti. In this field study in a mixed filarial (L. loa and M. perstans) endemic region of Gabon, 157 L. loa amicrofilaraemic blood samples (AMF; diagnosed by leucoconcentration followed by standard microscopic examination) from the residents from four villages were screened by the 15r3-nested PCR assay. The assay detected 106 occult infected subjects among the 157 AMF individuals (68%), including 59 of 87 adults (68%) and 47 of 70 children (67%). In each village the prevalence of occult infection was, respectively, 38%, 52%, 79% and 80% for Moyabi, Djoutou, N'djokaye and Okoumbi. The annual transmission potential (ATP) of loiasis has been estimated to be 250 infective larvae (L 3) per man per year for Moyabi and Djoutou, 1800 for N'djokaye and 433000 L3/man/year for Okoumbi. This implies a correlation between occult infection of loiasis and the intensity of transmission. By contrast, the prevalence of L. loa microfilariae was 21% for Okoumbi, 22%, for N'djokaye and 19% for Djoutou and Moyabi. These results show that the prevalence of loiasis in this region of Gabon is higher than previously described by standard microscopic examination and that the application of this assay will be significant in the development of control strategies for loiasis.

Accurate and specific diagnosis of human loiasis is of crucial importance in an endemic area where two-thirds of infected individuals are without circulating microfilariae (occult loiasis). By using the polymerase chain reaction (PCR) and specific primers to the repeat 3 region (15r3) of the gene coding for Loa loa 15-kDa polyprotein antigen, DNA was amplified from total blood lysate of occult-infected subjects. A 396-bp DNA fragment was specifically detected. We tested the specificity of this method by qualitative hybridization to PCR products using blood lysates of the following subjects: (1) from Gabon (80 individuals residing in L. loa endemic area where loiasis exists sympatrically with Mansonella perstans); (2) from Togo (12 individuals infected with Onchocerca volvulus and M. perstans); (3) from Tahiti (12 individuals infected with Wuchereria bancrofti); and (4) from Mali (12 individuals infected with O. volvulus and M. perstans). Samples from Gabon included 60 L. loa amicrofilaremics and 20 L. loa occult-infected subjects. Qualitative hybridization carried out at 50 degrees C on PCR products, using a 15r3-specific oligonucleotide probe, revealed hybridization with L. loa-infected samples from Gabon and four samples from Togo after 2 days exposure to the film. The positive samples from Togo were characterized by the use of nested PCR. Three nested PCR products have been sequenced. No differences were observed between the three sequences and they are 99.72% identical to L. loa 15r3. None of bancroftian-infected individuals from Tahiti, nor O. volvulus- and M. perstans-infected individuals from Mali reacted after 1 week's exposure (overexposure) to the film. This allows us to conclude first that our 15r3 PCR assay is specific for L. loa and secondly that L. loa infections occur in Togo. The sensitivity of this 15r3 PCR assay was further investigated with occult patients and field-collected amicrofilaremic samples. We found that 19 of the 20 occult-infected individuals were positive on Southern hybridization, whereas 35/60 amicrofilaremics were positive. These results have shown that the sensitivity of this assay in detecting unequivocal, parasitologically proven occult loiasis was 95%, while the specificity with regard to the sympatric M. perstans was 100%.

A polymerase chain reaction (PCR)-based method to detect Loa loa DNA in the blood lysate of infected individuals is described. A set of primers was designed to amplify the repeat 3 sequence (15r3) of the gene encoding a putative L. loa allergen. The qualitative PCR was carried out using blood lysates from subjects from an L. loaendemic area of Gabon where loiasis exists sympatrically with Mansonella perstans, and from individuals from a loiasis-free area in Togo infected concomitantly with M. perstans and Onchocerca volvulus. No specific amplification was observed after ethidium bromide staining of a gel containing M. perstans and O. volvulus control samples. In contrast, a 396-basepair (bp) DNA was detected in all L. loa microfilaremic individuals and in seven of the 20 L. loa amicrofilaremic subjects diagnosed by leukoconcentration. Qualitative Southern blots carried out at high stringency (65 degrees C) using 15r3 oligonucleotide probe revealed hybridization only with L. loa samples (5 of 5 microfilaremic individuals and 15 of 20 amicrofilaremic individuals), confirming the results obtained with ethidium bromide staining of PCR products. We conclude that this 396-bp sequence could be used as a species-specific diagnostic tool for occult loiasis in an endemic area with concurrent filarial infections.

A ladder antigen of Loa loa was identified on Western blots of all life cycle stages probed with loaisis sera. The smallest subunit has a relative M(r) of about 15 kDa and larger subunits represent size increments of 15.0 kDa. An 1800-bp genomic clone encoding this antigen was characterized further by restriction mapping. Southern blot analysis, and nucleotide sequencing. The antigen is encoded by multiple copies of a gene, linked in tandem repeats of 396 bp, each of which encodes 132 amino acids. These repeats have diverged sufficiently to produce distinct restriction enzyme sites and Southern blot hybridization patterns. The 1764-bp insert contains no introns and encodes 588 amino acids, representing one incomplete and four complete repeats. At the 3' end of three repeats, there are consensus proteolytic cleavage sites; one repeat has no cleavage site. Two perfect repeats show a 93.9% amino acid identity with one another; the rest of the repeats, despite being adjacent to one another, show only 31-42% identical amino acids. Putative asparagine N-linked glycosylation sites are expressed by only two of the repeats. Despite this structural diversity, each L. loa repeat showed homology to Ascaris suum allergen and the homologue protein described in Dirofilaria immitis and Brugia pahangi.

The specificity of IgG4 as marker of infection has been investigated. The study was based on two well defined clinical groups: amicrofilaremic individuals with verified ocular passage of adult worms of Loa loa, and microfilaremic patients. Both groups were compared to Africans not exposed to loiasis infection and to Europeans. The study revealed that there is no significant difference in the level of parasite-specific IgG4 between individuals with occult infection (i.e. amicrofilaraemic, but infected) and microfilaremic individuals, but there is a significant difference between L. loa infected individuals and Mansonella perstans exposed people. IgG4 of individuals exposed to L. loa infection recognised specific antigens in the molecular weight range 12-30 kDa. We conclude that the elevated level of filarial-specific IgG4 is therefore not dependent upon the presence of circulating microfilariae and that serology using homologous L. loa low molecular weight antigens, can facilitate specific diagnosis of occult loiasis in an endemic area with mixed filarial infections.

Immunologic analyses of sera from 47 selected individuals living in a mixed filariasis transmission zone in Gabon were carried out. Onchocerca volvulus, Loa loa, Mansonella streptocerca, and M. perstans are transmitted in this region. Based on parasitologic findings and age, the 47 individuals were stratified into four groups: microfilaria negative (Mf-) children (3-15 years old), Mf- adults (> 15 years old), microfilaria positive (Mf+) children and Mf+ adults. For descriptive purposes, the term microfilaria positive refers to individuals with skin and blood microfilariae. Antifilarial antibody titers were determined using an enzyme-linked immunosorbent assay with Dipetalonema viteae antigens. In general, children had higher titers of IgG antibodies than adults. For the IgG1, IgG2, and IgG3 subclass responses, both age and microfilarial status appeared to be important variables since Mf- children consistently had the highest titers whereas Mf- adults had the lowest titers. For the IgG4 antifilarial response, only the microfilarial status was an important variable. All Mf+ individuals had significantly higher levels of IgG4 antibody than Mf- individuals. Pooled sera of Mf- and Mf+ individuals reacted with similar O. volvulus antigens on Western blots. Control sera of individuals who did not reside in the study area, but who had single infections with L. loa or M. perstans, did not react with any O. volvulus antigens.

A Loa loa EcoRI genomic library in lambda gt11 was screened with 32P-labeled L. loa DNA and 1 repetitive clone, LL20, was isolated. An 800-bp Rsa I fragment of LL20, which is L. loa specific, was subcloned into pUC19 and the recombinant plasmid was designated pRsa4. While the 3.8-kb Eco RI fragment of LL20 cross-hybridized to other filarial DNA under low stringency conditions, the 800-bp fragment of pRsa4 was L. loa specific under the same conditions. Further characterization of the insert of pRsa4 was therefore carried out. Its lower limit of detection is 800 pg of L. loa genomic DNA, it has a low copy number (50-100) and an interspersed distribution in the genome. As a probe it does not distinguish between simian and human L. loa DNA. The nucleotide sequence contains 69% A + T and 31% G + C and shows no notable internal repeats.

Tropical pulmonary eosinophilia (TPE) is believed to result from extreme immediate hypersensitivity to microfilariae localized in the pulmonary vasculature of some persons with lymphatic filariasis. Female BALB/c mice repeatedly immunized by ip injection of Brugia malayi microfilariae become amicrofilaremic within 24 hr of iv parasite challenge, whereas non-sensitized control animals remain patent for greater than 72 hr. Immunized, but not control mice, develop peripheral blood and pulmonary eosinophilia (2,000 cells/mm3 and 65,000 cells/bronchoalveolar lavage, respectively). Serum and bronchoalveolar lavage filarial-specific IgG antibodies are greater in sensitized mice than in controls (ELISA absorbance values 20- and 10-fold higher, respectively). Serum IgE antibody levels are also greater (P less than 0.01) in immunized parasite-challenged mice than in controls (mean cpm 125I-labeled anti-mouse IgE bound to B. malayi antigen-coated Sepharose beads: 7,852 vs. 1,741, respectively). This model exhibits several of the major features of human TPE: amicrofilaremia, elevated levels of serum IgG and IgE antibodies to microfilariae, and blood and pulmonary eosinophilia. This model may be useful in the examination of the role of filarial antigen-specific lymphoid cells and antibodies in regulating the pathologic responses to microfilariae trapped in the lung.

The ability of the filarial nematode Loa loa to infect 2 species of primates was studied. The primate species selected were closely related to species known to be susceptible. A mandrill (Mandrillus sphinx) and 6 cynomolgus monkeys (Macaca fascularis) were infected by subcutaneous injection of third-stage larvae of human L. loa from Gabon. The mandrill developed microfilaremia with an estimated prepatent period of 147 days, but microfilariae were not detected in any of the cynomolgus monkeys. Thus, mandrills appear permissive to human L. loa, whereas cynomolgus monkeys are not. Serum antibody responses were examined on western blots of adult L. loa antigens. Preinfection sera from all animals gave no reactions, but, after infection, sera from cynomolgus monkeys reacted more intensely and with more antigens than mandrill sera. Antibodies were still detectable in cynomolgus monkeys 15 mo postinfection. These reactions were compared with those found using human infection sera. Reactions with the cynomolgus monkey sera resembled those found with resistant endemic and amicrofilaremic human sera.

Haemonchus contortus eggs were extracted from sheep feces and known numbers were added to helminthologically sterile bovine feces to provide samples with seven, 30 and 60 eggs per gram (epg). At 60 epg, dilution techniques (modified Cornell-McMaster and modified McMaster) tended to overestimate the number of eggs and more eggs were recovered (mean of 121 and 88% respectively) with these techniques than with centrifugal concentration procedures (modified Cornell-63% and Wisconsin- 69%). At 30 epg, all techniques were comparable (modified Cornell-McMaster 67%, modified McMaster 63%, modified Cornell and Wisconsin 64%). At 7 epg, the Wisconsin (61%), modified Cornell (60%) and Cornell-McMaster (94%) techniques were comparable and better than the modified McMaster technique (16%). At all levels of epg, the modified Cornell and Wisconsin techniques recovered eggs from 100% of the samples. The Cornell-McMaster and modified McMaster techniques recovered eggs from 90 and 100% of samples at 60 epg; 40 and 100% at 30 epg; and 21 and 11% at 7 epg. With a gravitational concentration procedure, the Standard Vial, no more than 16% of the eggs at any level of epg were recovered and at 7 epg eggs were recovered from only one-half of the samples. Five gravitational concentration techniques were assessed over 66 to 490 epg. The Ovassay, Fecalyzer and modified Standard Vial techniques were comparable in efficiency (28%, 25% and 24% respectively), but the Standard Vial technique was less efficient (11%). Introduced into diagnostic parasitology was the concept of predictive values which is the proportion of samples that a technique correctly identifies as being negative for parasite eggs. At 7 epg this was calculated to be zero for the modified Cornell-McMaster, modified McMaster and Standard Vial techniques and 100 for the Wisconsin and modified Cornell techniques.

Detergent solubilized extracts of 125Iodogen surface-labelled Loa loa microfilariae revealed a relatively simple profile of two strongly labelled molecules of 23 and 67 kDa for blood microfilariae and several strongly labelled molecules of 23, 40, 42-67 kDa for in vitro born microfilariae. In addition, there were other weakly labelled molecules which were resolved after prolonged autoradiographic exposure. Surface molecules of 28, 29, and 33 kDa were unique to blood microfilariae, a 14.4 kDa molecule was unique to in vitro born microfilariae and molecules of 23, 40, and 75-84 kDa were common to both forms of microfilariae. The profile of excretory-secretory products consisted of molecules of 14.4-198 kDa. Human albumin was a predominant component of surface molecules and excretory-secretory products from blood microfilariae. Immunoprecipitation with occult and microfilaremic loaiasis sera demonstrated that the 23 kDa surface molecule and excretory-secretory products of 14.4 and 33 kDa were only recognized by occult loaiasis sera whereas surface molecules of 40 and 75-84 kDa and excretory-secretory products of 28 and 67 kDa were recognised by both sera. Studies with heterologous sera demonstrated that with the exception of the 75-84 kDa antigens, all the L. loa microfilarial surface antigens contained epitopes which were restricted to filarial parasites. Further studies revealed that the 23 kDa antigen was a protein which contained neither asparagine-N-linked oligosaccharides nor interchain disulfide-linkages.

C3-opsonized zymosan particles (C3-zymosan) have been utilized as reagents for detection of C3 receptor bearing cells. However, in this communication we present evidence that C3-zymosan particles are not useful for this purpose in some species with certain macrophage populations because of non-specific binding of unopsonized zymosan.

Alveolar macrophages (AM) of rats infected with 3000 Nippostrongylus brasiliensis infective larvae for 2, 8 or 32 days (D2, D8 or D32 AM) quantitatively surpassed AM from uninfected rats in one or more of IgG- or C3-dependent phagocytosis indices, beta-D-glucuronidase release, or spontaneous release of thymocyte activating factor (interleukin-1, IL-1) and hepatocyte stimulation factor (HSF). These observations suggest that N. brasiliensis infection results in the activation of AM. We have reported previously that a greater proportion of AM from infected rats expressed C3 receptors and were helminthocidal in vitro in the presence of complement than normal AM which were not helminthocidal. The acquisition of the activated state by AM during infection may play a role in vivo lung resistance against migrating helminth parasites.

Using broncho-alveolar lavage, we have studied the cellular responses in the rat lung following primary and secondary infection with Nippostrongylus brasiliensis. During the primary infection, there was a biphasic increase in total broncho-alveolar leucocytes and in the absolute numbers of macrophages, neutrophils, eosinophils and lymphocytes. The first peak occurred on days 4-6, and the second peak occurred around day 16, after infection. During the secondary infection there was an anamnestic-like response by all cell types. These data suggest that the broncho-alveolar leucocyte responses to infection have an immunological basis and that in addition to the alveolar macrophage, neutrophils, eosinophils and lymphocytes may play a significant role in lung resistance against migrating helminth larvae.

Filarial infections induce a spectrum of disease in their natural hosts, and by correlating immunity found in individuals with their disease pattern, one may delineate non-pathogenic, protective mechanisms. Loa loa is causal of mild to moderate pathology, and it is unique among the human filaria in that adult worms are occasionally visible during subconjunctival migration. To study immune mechanisms controlling microfilaremia, sera from 15 subjects with amicrofilaremic occult loiasis (OL) were compared with sera from 10 subjects with microfilaremic loiasis (ML) microfilaremia, (greater than 4000/ml) for their reactions with living microfilariae (mf). An IFA was first used to detect antibodies able to bind to the surface of living L. loa mf. ML subjects either did not react (7/10) or reacted only very weakly (3/10). Highly reactive sera were found only in OL subjects; 7/15 gave very bright fluorescence, 5/15 gave moderate reactions, and 3/15 were negative. Most of these antibodies were of the IgG class. Sera from all subjects were also reacted with living mf in an antibody-dependent cellular adherence test using normal leukocytes. Sera that were strongly positive in IFA showed strong adherence and IFA-negative sera were non-reactive. To identify the Ag involved, mf were surface iodinated, detergent-extracted Ag were immunoprecipitated, and Mr was determined on SDS-PAGE. Several OL sera, all highly reactive in the above tests, precipitated a 23-kDa molecule with which all ML sea failed to react. Sera from a mandrill experimentally infected with L. loa also precipitated the 23-kDa Ag when taken post-patency. In conclusion, it appears that certain people who control L. loa microfilaremia have high levels of IgG antibodies that bind to a surface Ag of 23 kDa and are able to mediate cellular adherence.

Somatic antigens of Loa loa adult worms with molecular weights of 15-180 kDa were identified by Western blot analysis using sera from 3 categories of parasitologically and clinically defined subjects from a loiasis endemic zone. Sera of occult, amicrofilaremic (OL), and 'resistant' individuals with no clinical signs of infection (R) reacted with an antigen of 160 kDa; sera of highly microfilaremic individuals (ML) did not. ML sera strongly reacted with an antigen of 18 kDa which was recognized only weakly or not at all by OL and R sera. At higher dilutions, OL sera only reacted with antigens at 23 and 160 kDa and ML sera reacted with antigens at 18 and 23 kDa, whereas R sera reacted with antigens at 23, 42, 54, 70, 100, and 160 kDa. These data suggested that R sera contained a higher concentration of antibodies which reacted with denatured, nitrocellulose-bound antigens. The IgG4 isotype predominated for all groups of sera, while IgG3 antibody responses were observed only with R sera. IgG1 antibodies were seen in all groups but reacted with fewer antigens than IgG4 antibodies, and no IgG2 antibody responses were detected. Sera against Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus, and Dirofilaria immitis cross-reacted with somatic antigens greater than 70 kDa, whereas none reacted with Loa loa antigens less than 23 kDa.

Several variables in the Cornell-Wisconsin centrifugal flotation technique were studied using helminthologically sterile bovine feces to which known numbers of Haemonchus contortus eggs had been added. Neither mode of mixing (levigation versus conventional), volume (15-60 mL) of water used for making the feces water suspension nor specific gravity (1.20-1.33) of the sucrose flotation solution affected egg recovery. Optimal times for centrifugation at 264 x g of first the feces water and then the feces sucrose suspension were three and five minutes respectively. Under these conditions 62.6% of the eggs were recoverable and there was a linear relationship between the number of eggs recovered and those added to the feces. About 30% of the unrecovered eggs were found in the fecal debris retained on the strainer. About 5% of the unrecovered eggs were found in the supernatant discarded after the feces water centrifugation and also in the matrix of the viscous sucrose solution. Addition of the detergent Triton X-100 caused a decrease in egg recovery. False negatives were not encountered between 3 to 70 epg; at 1.44 epg there was only one in 14 samples. Optimum procedures for the technique are presented.

Detergent solubilized extracts of 125Iodogen surface labelled adult Loa loa revealed a relatively simple profile consisting of a strongly labelled molecule at 29-31 kDa and weakly labelled molecules at 14.5, 17, 21, 23, 34, 58, and 86 kDa. Residents of a L. loa endemic zone were assessed clinically and parasitologically and classified as microfilaremic, amicrofilaremic with documented ocular passage of adult worms, or 'resistant' subjects without any signs of infection. Sera from these subjects were used to identify L. loa adult surface antigens. All 'resistant' sera immunoprecipitated the 29-31 kDa antigen although some were more strongly reactive than others. The amicrofilaremic sera strongly immunoprecipitated the 29-31 kDa antigen, whereas microfilaremic sera reacted weakly or not at all with this antigen. Longer exposures of immunoprecipitates of strongly reactive sera revealed the recognition of additional antigens of 86, 44, 34, 23, 21, 17 and 14.5 kDa. Studies with heterologous sera demonstrated that these antigens contain cross-reactive epitopes which are restricted to filarial parasites. Biochemical characterization of the predominant 29-31 kDa antigen showed that it bound concanavalin A, was sensitive to proteases, and its antigenicity was resistant to heat but sensitive to periodate and endo-beta-N-acetylglucosaminidase H. These observations suggest that it is a glycoprotein containing mannose and N-acetylglucosamine residues and that the carbohydrate moiety is important for antibody binding. The importance of the 29-31 kDa glycoprotein in the immunobiology of loaiasis is suggested by the finding that resistant and infected amicrofilaremic individuals have strongly reactive IgG antibodies to this antigen.

The objectives of the present study were to identify and characterize biochemically the major antigens of Brugia malayi microfilariae, a filarial parasite that infects humans. IgG antibodies in sera of mice which had cleared parasites from the bloodstream reacted with 30, 55, 94 and 150 kDa molecules of living microfilariae radioiodinated by the Iodo-bead method. Sera of humans infected with the related filariae Wuchereria bancrofti, Loa loa or Onchocerca volvulus immunoprecipitated molecules of similar size as well as two additional proteins of 22 and 43 kDa. Sera of uninfected North Americans or mice infected with Trichinella spiralis or Schistosoma mansoni did not recognize these radioiodinated antigens. Experiments to examine the possible surface localization and metabolism of these antigens showed that they were removed from intact parasites exposed to chymotrypsin or trypsin and that immunogenic molecules of 30, 55, and 150 kDa were released into excretory-secretory products by viable microfilariae. [35S]Methionine biosynthetically labeled polypeptide antigens of 22, 30, 35 and 150 kDa were detected by antibody reacted with intact microfilariae and/or their excretion products. Antigens of 30, 55, and 150 kDa appear to be glycoproteins as they bound wheat germ agglutinin and were biosynthetically labeled with [14C]N-acetyl-D-glucosamine. These data suggest that the surface of B. malayi microfilariae is a dynamic structure which synthesizes and sheds antigens.

Immunological and biochemical studies on some parasite species are hampered by a limited availability of parasite material. Thomas Egwang Jean-Paul Akue and Margaret Pinder discuss a technique for preserving material from adult filarial worms during transport through endemic areas.