Serological survey and risk factors of Aelurostrongylus abstrusus infection among owned cats in Italy.

Feline lungworms affect the respiratory tract of domestic cats causing respiratory conditions of various degrees. In this study, we investigated the exposure of cats to feline lungworm infections by detecting antibodies in a large population of animals from several regions of Italy. Sera of 1087 domestic cats living in regions of the north (n = 700), the centre (n = 227) and the south (n = 160) of Italy were examined by a newly developed indirect ELISA conceived for detection of antibodies against the most frequently occurring feline lungworm Aelurostrongylus abstrusus. Individual cat data (i.e., age, sex, neutering status and provenience) were analysed as potential risk factors for exposure to lungworm infections. Samples were additionally screened for feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) proviral DNAs. Overall, 9% (98/1087; 95% confidence interval (CI) 7.4-10.9%) of the animals tested seropositive to lungworm antibodies. Positive cats were identified in the north (7.1%; CI 5.5-9.3%), in the centre (5.3%; CI 3.0-9.0%) and in the South (22.5%; CI 16.7-29.6%), with more seropositive animals in the latter area (p < 0.05). The risk of lungworm infection in cats was significantly associated with age less than 6 months (i.e. 24.4%, p < 0.05) and FIV infection (p < 0.05). This large-scale serological survey confirms the exposure of cats to lungworm infections in Italy and that serological tests can be used to assess the distribution of lungworm infections in large populations of animals.

Seroprevalence, biogeographic distribution and risk factors for Aelurostrongylus abstrusus infections in Swiss cats.

The metastrongyloid nematode Aelurostrongylus abstrusus is a worldwide occurring feline lungworm. The spectrum of clinical signs in infected cats ranges from mild (e.g. nasal discharge or cough) to severe respiratory distress. The aim of this seroepidemiological study was to define prevalence and risk factors for A. abstrusus infections in Swiss cats, to assess the biogeographic distribution and to investigate the influence of temperature and altitude on the occurrence of this parasite. Sera of 4067 domestic cats were collected from all over Switzerland, tested for the presence of antibodies against A. abstrusus by a novel ELISA and the results correlated with biogeographic aspects. A subsample of 1000 datasets was used for risk factor analyses. Overall, 10.7% (434/4067, 95% confidence intervals [CI]: 9.7-11.7%) of the cats were tested positive, with variations from 0.0% to 20.0% among ten different biogeographic regions. Differences were significant between the Western (13.9%, CI: 11.4-16.7%) and the Eastern (9.2%, CI: 8.0-10.5%) Swiss Plateau, possibly attributable to the suitability of the areas for intermediate hosts. In total 90.3% (392/434) of the seropositive cats originated from regions lower than 700 m above sea level. Correspondingly, 98.9% (429/434) of positive samples were obtained from regions with a mean temperature higher than -2 °C in January, suggesting altitude and temperature being limiting factors for A. abstrusus infections in Switzerland. Concerning individual risk factors, prevalence was higher in intact (15.5%, CI: 9.5-23.4%) than in neutered cats (5.8%, CI: 7.9-10.4%). Young adult cats (aged 11-22 months) were significantly more often seropositive (10/76, 13.2%, CI: 6.5-22.9%) than kittens aged 1-10 months (1/34, 2.9%, CI: 0.1-15.3%) or adult and senior cats > 22 months (58/889, 6.5%, CI: 5-8.4%). Outdoor cats and cats presenting respiratory signs tend to be more often positive than indoor cats (p = 0.077) and animals without respiratory signs (p = 0.086), respectively. We here confirm that the use of a serological test can contribute to improve the identification of infected animals, through evaluation of risk factors on a population level and for a better management on an individual level, overcoming the challenges represented by faecal examinations and the correlated underestimation of the occurrence of A. abstrusus in cats.

Detection of specific antibodies in cats infected with the lung nematode Aelurostrongylus abstrusus.

Feline aelurostrongylosis, caused by the metastrongylid nematode Aelurostrongylus abstrusus, is an underestimated respiratory parasitosis. Its diagnosis currently mainly relies on the isolation of first stage larvae from fresh faecal samples. The aim of our study was to develop a serological test for the detection of specific antibodies against A. abstrusus by ELISA. We used recombinant major sperm protein (MSP) of the bovine lungworm Dictyocaulus viviparus as detection antigen and evaluated two different ELISA plates (Maxisorp and Immobilizer™ Amino-plate, Nunc Roskilde, Denmark) with two different enzyme systems [alkaline phosphatase (AP) and horseradish peroxidase (HRP)]. Sera from cats experimentally (n=54) and naturally (n=17) infected with A. abstrusus and from randomly selected cats with different medical issues (n=160) were used to determine sensitivity and specificity. Furthermore, cross-reactions were evaluated using sera from cats naturally (n=71) and experimentally (n=8) infected with different nematodes. A sensitivity of 100% was obtained with sera from experimentally infected cats at 10 weeks post infection using MSP on the Immobilizer™ Amino-plate with HRP, while it ranged between 90.5 and 95.2% in the other ELISA set-ups. Using sera from naturally infected cats, a sensitivity of 88.2% (95% confidence interval: 63.6-98.5%) was achieved in all four set-ups. The specificity was 85.2-94.4% in sera from uninfected cats prior to experimental infection and 68.1-90% in randomly selected cats depending on the plate and enzyme system. The number of seropositive cats increased over time post infection. Serological follow-up showed a decrease of antibody levels within 30days after anthelmintic treatment. Seropositive reactions were observed with sera from stray cats naturally infected with Toxocara cati, Capillaria sp., hookworms and Taeniidae; however, coproscopic false negative A. abstrusus findings cannot be excluded. The serological detection of specific antibodies against A. abstrusus using ELISA requires a single serum sample and therefore represents a valid alternative for reliable individual diagnosis of A. abstrusus in cats and facilitates mass screening, overcoming the usually difficult collection of cat faeces.

A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals.

BACKGROUND: Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is desirable for monitoring health of seals in the wild and in captivity. Previously, an ELISA based on recombinant bovine lungworm major sperm protein (MSP) as diagnostic antigen was developed for lungworm diagnosis in cattle. In the present study, this test was adapted for detection of antibodies against lungworms in harbour (Phoca vitulina) and grey seals (Halichoerus grypus). Furthermore, sera of northern elephant seals (Mirounga angustirostris) were tested to evaluate whether the harbour/grey seal ELISA is suitable for this seal species as well. METHODS: For ELISA evaluation, lungworm-positive and -negative sera of harbour and grey seals were analysed using horseradish peroxidase (HRP)-conjugated Protein A as secondary antibody. Optical density was measured and a receiver operating characteristic (ROC) analysis was performed to determine a cut-off value. Potential cross-reactions were examined by testing serum of seals positive for gastrointestinal and heart nematodes, but negative for lungworm infections. In addition, sera of northern elephant seals were analysed. RESULTS: Harbour and grey seal serum samples showed significant differences in optical density (OD) between serum of infected and uninfected animals resulting in a cut-off value of 0.422 OD with a specificity of 100% (95% CI: 87.23-100%) and a sensitivity of 97.83% (95% CI: 88.47-99.94%). Cross-reactions with heart or gastrointestinal nematodes were not observed. Analysis of northern elephant seal samples resulted in detection of antibodies in animals positive for lungworm larvae at faecal examination. CONCLUSIONS: The ELISA presented is a valuable method for detection of lungworm infections in live harbour and grey seals, providing a monitoring tool to reveal epidemiological dynamics of lungworm infections during health surveillance in free-ranging seals. Furthermore, ELISA results may aid institutions with harbour and grey seals under human care on decisions regarding anthelminthic treatment of individual animals.

Analysis of heavy-chain antibody responses and resistance to Parelaphostrongylus tenuis in experimentally infected alpacas.

The parasitic nematode Parelaphostrongylus tenuis is an important cause of neurologic disease of camelids in central and eastern North America. The aim of this study was to determine whether alpacas develop resistance to disease caused by P. tenuis in response to a previous infection or a combination of controlled infection and immunization. Alpacas were immunized with a homogenate of third-stage larvae (L3) and simultaneously implanted subcutaneously with diffusion chambers containing 20 live L3. Sham-treated animals received adjuvant alone and empty chambers. The protocol was not effective in inducing resistance to oral challenge with 10 L3, and disease developed between 60 and 71 days following infection. Immediately following the onset of neurologic disease, affected animals were treated with a regimen of anthelmintic and anti-inflammatory drugs, and all recovered. One year later, a subset of alpacas from this experiment was challenged with 20 L3 and the results showed that prior infection induced resistance to disease. Primary and secondary infections induced production of conventional and heavy-chain IgGs that reacted with soluble antigens in L3 homogenates but did not consistently recognize a recombinant form of a parasite-derived aspartyl protease inhibitor. Thus, the latter antigen may not be a good candidate for serology-based diagnostic tests. Antibody responses to parasite antigens occurred in the absence of overt disease, demonstrating that P. tenuis infection can be subclinical in a host that has been considered to be highly susceptible to disease. The potential for immunoprophylaxis to be effective in preventing disease caused by P. tenuis was supported by evidence of resistance to reinfection.

Construction of a complementary DNA library for Parelaphostrongylus tenuis and identification of a potentially sero-diagnostic recombinant antigen.

Newly developed serological tests for diagnosing parelaphostrongylosis in cervids, using the excretory-secretory products (ES) of the infective larvae of Parelaphostrongylus tenuis in enzyme-linked immunosorbent assays (ELISAs), have demonstrable superiority over the traditional method of larval recovery and microscopic identification. To generate a source of ELISA antigen by genetic engineering, we created a complementary DNA (cDNA) expression library by the reverse transcription of mRNA of P. tenuis adult worms, and ligation with the vector lambda-ZAP II. The library was screened using antisera produced in mice by immunization with a somatic antigen preparation of adult worms. Seventeen clones were isolated, sequenced, and checked for similarity to other DNA sequences in GenBank. A previously identified parasite gene encoding an aspartyl protease inhibitor (API) was isolated from the cDNA library, subcloned and expressed using the pET expression vector to produce a glutathione S transferase (GST)-His-S.Tag-P. tenuis API fusion protein (molecular weight = 63 kDa). An enzyme-linked immunosorbent assay utilizing the API fusion protein as the coating antigen was used to serologically diagnose all white-tailed deer (WTD, 10 out of 10) that had been inoculated with 6 - 150 L3 P. tenuis, indicating that the antigen may be a useful serodiagnostic antigen for P. tenuis infection in this cervid species.

Immunoglobulin responses of northern elephant and Pacific harbor seals naturally infected with Otostrongylus circumlitus.

Immunoglobulin (Ig) binding patterns of Pacific harbor seals (PHS, Phoca vitulina richardsi) and northern elephant seals (NES, Mirounga angustirostris) to tissues of adult Otostrongylus circumlitus were examined by immunoblotting to investigate the role of age in the unusual response of juvenile NES to infection with O. circumlitus. Serum was taken from NES between March 1997 and March 2001 and from PHS between May 1996 and August 1999. The serum of seals infected with O. circumlitus contained antibodies that bound to all nematode tissues examined. Intensity of band staining on Western blots suggested that there were higher levels of antibody recognizing the excretory-secretory (ES) glands in the serum of NES that were 1 yr and older and in the majority of PHS compared with that in 2- to 9-mo-old NES. All juvenile NES infected with O. circumlitus and a proportion of the PHS and older NES infected with O. circumlitus contained Ig specific to a 28 kDa protein band that was dominant in the female reproductive tract of the nematode. The Ig binding patterns of NES and PHS to adult Parafilaroides sp., larval Pseudoterranova sp., and larval and adult Anisakis sp. differed sufficiently from that of O. circumlitus that immunoblotting for the 28 kDa protein could be useful for diagnosis of this parasite in juvenile NES. The banding patterns suggest that O. circumlitus nematodes die and disintegrate in PHS and NES and that NES of 1 yr and older and most PHS respond differently to the ES glands than 2- to 9-mo-old NES.

Establishment of adult Parelaphostrongylus tenuis, patent infections, and acquired immunity after experimental infection of white-tailed deer (Odocoileus virginianus) and red deer (Cervus elaphus elaphus).

Experimental Parelaphostrongylus tenuis infections were established in white-tailed deer (Odocoileus virginianus) and an atypical host, red deer (Cervus elaphus elaphus). Groups of deer were fed 10, 25, or 100 third-stage larvae (L3) of P. tenuis and received a single equivalent challenge exposure at varying intervals. Infections were monitored up to 6 yr in white-tailed deer and up to 2.8 yr in red deer. The prepatent period in white-tailed deer varied from 91 to 1,072 days (381 +/- 374) and in red deer from 105 to 358 days (167 +/- 77). Adult worms lived for up to 6 yr in white-tailed deer. Although most had patent infections until necropsy, latent periods were observed regardless of season. Adult worms lived for up to 2.8 yr in red deer, and patent infections persisted for 20-363 days (152 +/- 106). Patent infections were correlated with the presence of adult worms in blood vessels and sinuses of both deer species. Worms were restricted to the subdural space in all deer with latent and occult infections. Adult worm recovery in white-tailed deer fed 10 or 25 L3 corresponded to the mean intensities reported in natural infections of white-tailed deer Recovery from deer fed 100 L3 was not typical of natural infection intensities. Adult P. tenuis established in all groups of red deer, but neurologic disease was restricted to animals fed 100 L3. Acute neurologic disease was associated with subdural hemorrhage and occurred at 11 mo postinfection in 2 red deer. The absence of postchallenge patent periods and the persistence of occult infections indicated that challenge exposures did not establish. These data indicate that acquired immunity to P. tenuis was established by 6 mo postinfection in both white-tailed and red deer. Latent periods in white-tailed deer and latent infections in red deer reinforce the need for a reliable diagnostic assay.

Immunodiagnosis of experimental Parelaphostrongylus tenuis infection in elk.

Elk infected with the meningeal worm, Parelaphostrongylus tenuis (Protostrongylidae), do not consistently excrete larvae in feces, making the current method of diagnosing live animals using the Baermann fecal technique unreliable. Serological diagnosis could prove more useful in diagnosing field-infected animals but depends on the identification and availability of good quality antigen. To mimic field infections, 2 elk were inoculated with 6 infective L3 larvae of P. tenuis, and another 2 with 20 L3 larvae. Fecal samples were examined for nematode larvae using the Baermann technique and serum samples taken were tested for anti-P. tenuis antibody with ELISAs by using the excretory-secretory (ES) products of L3, and sonicated adult worms as antigens. One animal passed first-stage larvae in its feces 202 days postinoculation, but passed none thereafter. The remaining 3 inoculated animals did not pass larvae. In contrast to parasite detection, antibodies against larval ES products were detected in all animals starting from 14 to 28 days postinoculation and persisted until the termination of the experiment on day 243 in 2 animals that harbored adult worms. Antibodies against somatic antigens of the adult worm were not detected until day 56 but also persisted until the end of the experiment in the animals with adult worms. In 2 elk that had no adult worms at necropsy, anti-ES antibodies were detected transiently in both, while anti-adult worm antibodies were present transiently in one. These findings confirm the superiority of P. tenuis larval ES products over somatic adult worm antigens as serodiagnostic antigens, as previously observed in studies of infected white-tailed deer, and extend the application of the newly developed ELISA test in diagnosing and monitoring cervids experimentally infected with P. tenuis.

Detection of anti-parelaphostrongylus tenuis antibodies in experimentally infected and free-ranging moose (Alces alces).

Confirming Parelaphostrongylus tenuis infection in moose (Alces alces) and other susceptible hosts is difficult. An enzyme-linked immunosorbent assay (ELISA) was developed using the excretory-secretory (ES) products of third-stage P. tenuis larvae (ES-ELISA) and the test applied to serum samples obtained from seven moose calves (5-9.5 mo old) given infective larvae (L3) in doses approximating those likely to be received in nature (3-30 L3). Anti-P. tenuis immunoglobulin G antibodies were detected in all seven inoculated moose during the course of infection until the termination of experiment 61-243 days post-inoculation (DPI). Five animals tested between 16-25 DPI had significant antibody levels, while a sixth animal did not test positive until 46 DPI. The seventh animal was not tested until 199 DPI. Antibody levels remained elevated in all five animals that harbored adult worms at the termination of the experiment. Whereas, antibody levels showed a gradual decline in the two remaining animals, presumably because of death of worms, and antibodies were undetected in one animal at the time of necropsy. The other animal displayed an anamnestic increase in antibody level following a challenge inoculation of infective larvae. Terminal and peak optical density (OD) values detected by ES-ELISA strongly correlated with inoculation dose (r = 0.98, P = 0.02 and r = 0.95, P = 0.04, respectively) among animals harboring adult worms (n = 4) but not significantly with the number of worms recovered postmortem (peak OD, r = 0.82, P = 0.18; terminal OD, r = 0.93, P = 0.07). Unlike the ES products, use of somatic antigens of the adult worm in ELISA did not provide satisfactory results. Antibodies to P. tenuis were detectable by ES-ELISA in two of 21 free-ranging moose from an enzootic area but not from any of 23 animals from a non-enzootic area. The ES-ELISA appears to be a useful test for assessing exposure of moose to P. tenuis.

Serological diagnosis of Parelaphostrongylus tenuis infection in white-tailed deer and identification of a potentially unique parasite antigen.

Serological diagnosis of Parelaphostrongylus tenuis infection should offer many advantages over the currently used method of fecal analysis that relies on a patent infection. Toward this end, we investigated the presence of P. tenuis-specific antibodies in experimentally infected white-tailed deer (WTD) and of unique P. tenuis antigens that may be exploited for serodiagnosis. WTD infected with 6, 20 or 100-150 P. tenuis third-stage larvae (L3) had anti-parasite antibodies from as early as 21 days postinoculation (dpi) until the end of the experiment (147 dpi). Peak anti-P. tenuis enzyme-linked immunosorbent assay (ELISA) titers in individual animals ranged from 1:70 to 1:5,700. Serum from infected WTD reacted with 5 distinct P. tenuis L3 antigens (105, 45, 37, 32, and 19 kDa) as detected by the immunoblotting technique. Serum from caribou infected with Parelaphostrongylus andersoni or Elaphostrongylus rangiferi reacted with all antigens except the 37-kDa antigen of L3, indicating that it may be unique to P. tenuis and can serve as a serodiagnostic antigen. The 37-kDa antigen appears to be present in the adult P. tenuis but not adult E. rangiferi or E. cervi. The development of an ELISA utilizing the unique antigen of P. tenuis should lead to a reliable diagnostic assay for P. tenuis infection in WTD.

Meningeal worm evokes a heterogeneous immune response in elk.

Meningeal worm (Parelaphostrongylus tenuis) is a neurotropic nematode of ungulates in eastern North America. Lack of an effective diagnostic test increases the concern of translocating potentially infected ungulates into western North America, where P. tenuis does not occur naturally. In an attempt to identify serodiagnostic molecules, we determined (1) whether elk (Cervus elaphus) experimentally infected with P. tenuis produce antibodies against infective larvae or adult worms, and (2) if sera consistently recognize antigens that distinguish P. tenuis from a common nematode parasite of elk, the lungworm Dictyocaulus viviparus. Each of 10 elk were exposed to 15 or 300 infective P. tenuis larvae. Serum was collected (0, 41, and 83 days post-exposure and at necropsy) and monitored for antibodies using the enzyme-linked immunosorbent assay (ELISA) and immunoblot. When reactivity of sera with larval P. tenuis protein was compared (day 0 versus 83), ELISA values were significantly higher on day 83 for elk exposed to 15 or 300 parasites. Likewise, ELISA values using protein of adult P. tenuis were higher for elk exposed to 300 larvae. Immunoblots showed that sera from elk, with adult worms in the central nervous system, consistently recognized the 25-27, 28-30, and 34-36 kDa antigens of infective larvae after 83 days. However, many D. viviparus molecules were found to cross-react with antibodies formed against meningeal worm antigens. Use of adult worm proteins for serodiagnosis appears limited, because no protein was consistently recognized by sera collected from elk exposed to 15 larvae. We believe that development of a reliable diagnostic test for meningeal worm requires more research addressing cross-reactivity and detection of P. tenuis during the incubation stage.

Bronchoalveolar cellular responses of goats following infections with Muellerius capillaris (Protostrongylidae, Nematoda).

The development of Muellerius capillaris in the lung of goats was associated with marked tissue damage and pronounced a cellular reaction. Using broncho-alveolar lavage, the time course of the cellular responses was studied following primary and secondary infection. During the primary infection, there was a biphasic increase in total broncho-alveolar leucocytes (an average of 294.0 +/- 137.0 cells microl[-1]) and in the absolute number of macrophages (182.0 +/- 82.0 cells ul[-1]), lymphocytes (68.5 +/- 35.0 cells microl[-1]), eosinophils (35.3 +/- 16.4 cells microl[-1]) and neutrophils (10.9 +/- 8.7 cell microl[-1]). The lung tissue reaction against worms consisted of a mild infiltration of inflammatory cells. The secondary infection resulted in significant changes in the pulmonary tissue characterised by severe inflammation, leading to widespread granulomatous formation throughout the parenchyma, hyperplasia of cells Type II and a leucocytosis in the broncho-alveolar fluids, with an anamnestic-like response by all cell types. The overall average of the total leucocytes, macrophages, lymphocytes, eosinophils and neutrophils was 529.3 +/- 347.4; 265.4 +/- 148.1; 127.3 +/- 100; 125.4 +/- 100.1 and 14.0 +/- 8.7 cells microl(-1), respectively. Secondary infection also resulted in 56% reduction of worms established in the lungs and 72.3% of L1 larval production. These data suggest that the broncho-alveolar leucocyte response to infection has an immunological basis and that the alveolar macrophages, neutrophils, eosinophils, and lymphocytes may play a significant role in lung resistance against protostrongylid nematodes.

Antigens of adults and third-stage larvae of the meningeal worm, Parelaphostrongylus tenuis (Nematoda, Metastrongyloidea).

We studied the antigens of adults and third-stage larvae of the meningeal worm, Parelaphostrongylus tenuis, in an attempt to identify potential serodiagnostic molecules for this important infection of wild ungulates. Soluble extracts of P. tenuis adult worms and third-stage larvae were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2D) electrophoresis and analyzed by immunoblotting using purified rabbit anti-P. tenuis immunoglobulin G (IgG). The IgG antibodies were obtained from animals immunized with P. tenuis adult worm or third-stage larva soluble extract and serum from elk infected with P. tenuis. Out of more than 75 antigens (as shown by 2D electrophoresis and immunoblotting), 7 antigens from adults (four 170-120-kD molecules with isoelectric points between 6.0 and 6.6, two 55-kD molecules with isoelectric points of 5.6 and 5.8, and one 13-kD molecule) and 2 antigens from third-stage larvae (one 25-30-kD molecule with an isoelectric point of 6.3 and one 13-kD molecule) distinguished P. tenuis from two other nematodes, Dictyocaulus viviparus and Trichinella spiralis. Initial results using serum from experimentally infected elk indicate that this serum recognized a similar profile of P. tenuis antigens when compared with the serum from immunized rabbits. This research has set the foundation for the development of a test for P. tenuis infections in wild and recently domesticated elk and and other ungulates.

[Histopathologic study of the host-parasite relationship: the earthworm-, wild boar-metastrongyle model]. / Etude histo-pathologique de la relation hôtes-parasite: le modèle lombric, sanglier-métastrongles.

A histopathological study was conducted on two hosts of metastrongyles (Metastrongylus sp.): earthworms and wild boars (Sus scrofa Linnaeus, 1758). In the earthworm intermediate host there were rare granulomas outside the normal site occupied by larvae (blood sinuses of calciferous glands). Given the small number of these reactions, they could not constitute a limiting factor for parasitism in this host. However, the lungs of wild boars contained numerous lesions associated with the presence of these nematodes. Such lesions, often inflammatory, were part of an immunological defence mechanism which controls the extent of parasitism in wild boar.

Differences in responses of rat- and guinea-pig-eosinophils to eosinophil chemotactic factors derived from Angiostrongylus cantonensis.

Eosinophil chemotactic activity associated with whole worm extracts of the young adult worms (YA) and 1st stage larvae (L1) of Angiostrongylus cantonensis was assessed using guinea-pig- and rat-eosinophils. Both whole worm extracts were potently chemotactic to guinea-pig-eosinophils whereas only the whole worm extract of L1 was chemotactic to rat-eosinophils. Gel filtration chromatography of YA-whole worm extract yielded an eosinophil chemotactic factor (ECF-YA) with an estimated molecular weight of 16,900. ECF-YA was resistant to heating and pronase digestion but sensitive to periodate oxidation, suggesting that chemotactic activity was possibly associated with the sugar portion of the glycoprotein molecule. Guinea-pig- and rat-eosinophils were deactivated by previous incubation with homologous whole worm extracts but not with heterologous ones. When guinea-pig-eosinophils were treated with trypsin or pronase, their chemotaxis to ECF-YA was significantly inhibited, and pronase treatment was more effective. Both deactivated and trypsin-treated guinea-pig-eosinophils completely recovered their chemotaxis responses after in vitro culture for 12 and 24 h, respectively. When those eosinophils were cultured in vitro in the presence of puromycin or cycloheximide, however, their chemotaxis responses could not be recovered. These data clearly indicate that guinea-pig-eosinophils probably possess a kind of receptor (or 'recognition unit') capable of reacting to ECF-YA, and also that the receptor may be protein or glycoprotein molecules, and reproducible.

Immunoglobulin G-dependent classical complement pathway activation in neutrophil-mediated cytotoxicity to infective larvae of Angiostrongylus cantonensis.

The participation of antibody and complement in cell-mediated adherence and cytotoxicity to infected larvae (L3) of Angiostrongylus cantonensis was investigated in vitro. Of the different cell types involved in the reaction, neutrophils were seen to have a predominant role in immune serum--dependent adherence and cytotoxicity to L3. In the presence of immune serum, cytotoxicity to L3 by neutrophils from infected rats was twice that of neutrophils from normal rats. Although mononuclear cells and eosinophils from infected rats significantly increased the adherence to L3, they had little lethal effect on L3. A further study using gel filtration (Sephacryl S-200) and affinity chromatography (protein A) revealed that immunoglobulin G (IgG) alone was responsible for the complement activation in neutrophil-mediated killing of L3. Neither adherence nor cytotoxicity to L3 by neutrophils were affected when immune serum was heated to 50 degrees C or treated with zymosan, but they were markedly decreased when immune serum was treated with Mg2(+)-ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results of this study indicate that the neutrophil-mediated adherence and cytotoxicity to L3 of A. cantonensis are mediated through IgG-dependent classical complement pathway activation.

Acquired immunity in rats against Angiostrongylus cantonensis infection.

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).

Influence of immune treatments against Dirofilaria immitis infection in dogs.

Mongrel dogs were inoculated with two kinds of antigenic substances. The first was a phosphate buffered saline extract of whole Dirofilaria immitis mixed with aluminum hydroxide gel (group 1), and the second was an orally administered live Metastrongylus apri infective larvae (L3) (group 2). Both groups were then infected with D. immitis L3. Indirect hemagglutination (IHA) tests showed that the antibody was produced by these inoculations before the infection was introduced, even in dogs inoculated with M. apri. This suggests a cross-reactivity between D. immitis and M. apri. The initial passive cutaneous anaphylaxis (PCA) antibody production was markedly delayed by about 70 days in group 2 compared with the production in the infected control dogs (group 3). The appearance of microfilaremia was also delayed by about one month in group 2 compared with that in the above control group. All dogs were sacrificed after the termination of the observation and worms recovered from the right ventricle and pulmonary arteries were counted and measured. The results indicated that immunization resulting from the homologous worm-somatic antigen might accelerate the growth of the infected larvae, whereas immunization resulting from the heterologous worm antigen, but cross-reactive to D. immitis, might disadvantageously affect the growth.

Humoral immune response of Balb/c mice to larval and adult worm antigens of Angiostrongylus malaysiensis.

The development of Angiostrongylus malaysiensis in Balb/c mice and the humoral response due to it were studied by using the enzyme-linked immunosorbent assay (ELISA) with adult worm and L3 antigens. The worms recovered from mice were seen in the brain tissue only, they failed to migrate to the lung as in the normal host (rats). The antibody titres of sera from infected mice, showed similar patterns in response to L3 antigen and to adult worm antigen. However, the highest antibody response could be detected by L3 antigen in the early period after infection while the adult worm antigen detected a higher response in the later stages of development.