Analysis of Toxocara canis larval excretory-secretory antigens: physicochemical characterization and antibody recognition

Toxocara canis larval excretory-secretory antigens (TEX) were resolved by gradient pore polyacrylamide gel electrophoresis and analyzed using silver, periodic acid-Schiff, and immunoperoxidase stains. At least 15 bands between 29 and 94 kilodaltons (kDa) were detected by silver stain, all of which were recognized by antibodies in serum of a patient with visceral larva migrans. Immunoperoxidase stain detected an additional band at 92 kDa and 4-6 others above 200 kDa. Periodic acid-Schiff stain also detected the high molecular weight components, but did not detect constituents of approximately 53 and 57 kDa. Immunoperoxidase stain using antibody from the vitreous fluid of an ocular larva migrans patient detected 2 TEX components, approximately 76 and 80 kDa. Antigens were compared with respect to batch of larvae and age of larvae in culture. Qualitative differences that correlated with batch were found in the number of constituents above 200 kDa, and in 1 component of 78 kDa. Qualitative differences were noted in many minor components, some of which appeared to correlate with age of larvae in culture. Major TEX constituents were recognized consistently by antibody, regardless of batch or age of larvae. Total protein production per larva was approximately 8 ng/day, and was consistent over time. There was no evidence of neutral proteases in TEX.     

The mitochondrial genome of Baylisascaris procyonis

Background

Baylisascaris procyonis (Nematoda: Ascaridida), an intestinal nematode of raccoons, is emerging as an important helminthic zoonosis due to serious or fatal larval migrans in animals and humans. Despite its significant veterinary and public health impact, the epidemiology, molecular ecology and population genetics of this parasite remain largely unexplored. Mitochondrial (mt) genomes can provide a foundation for investigations in these areas and assist in the diagnosis and control of B. procyonis. In this study, the first complete mt genome sequence of B. procyonis was determined using a polymerase chain reaction (PCR)-based primer-walking strategy.

Methodology/Principal Findings

The circular mt genome (14781 bp) of B. procyonis contained 12 protein-coding, 22 transfer RNA and 2 ribosomal RNA genes congruent with other chromadorean nematodes. Interestingly, the B. procyonis mtDNA featured an extremely long AT-rich region (1375 bp) and a high number of intergenic spacers (17), making it unique compared with other secernentean nematodes characterized to date. Additionally, the entire genome displayed notable levels of AT skew and GC skew. Based on pairwise comparisons and sliding window analysis of mt genes among the available 11 Ascaridida mtDNAs, new primer pairs were designed to amplify specific short fragments of the genes cytb (548 bp fragment) and rrnL (200 bp fragment) in the B. procyonis mtDNA, and tested as possible alternatives to existing mt molecular beacons for Ascaridida. Finally, phylogenetic analysis of mtDNAs provided novel estimates of the interrelationships of Baylisasaris and Ascaridida.

Conclusions/Significance

The complete mt genome sequence of B. procyonis sequenced here should contribute to molecular diagnostic methods, epidemiological investigations and ecological studies of B. procyonis and other related ascaridoids. The information will be important in refining the phylogenetic relationships within the order Ascaridida and enriching the resource of markers for systematic, population genetic and evolutionary biological studies of parasitic nematodes of socio-economic importance.     

Distribution, prevalence, and genetic characterization of Baylisascaris procyonis in selected areas of Georgia

Baylisascaris procyonis is an intestinal nematode of raccoons (Procyon lotor) that can cause fatal larval migrans in numerous species of birds and mammals, including humans. Although this parasite has historically been absent in the southeastern United States, it has been found in isolated regions in the Appalachian Mountains and was recently documented in DeKalb County, Georgia. The first objective of the current study was to investigate the distribution and prevalence of B. procyonis in selected populations of raccoons in Georgia. Intestinal tracts of 312 raccoons from 25 Georgia counties were examined for B. procyonis. The only county where B. procyonis was detected was Clarke County, where 12 of 116 (10.3%) raccoons were infected. In Clarke County, significantly more juveniles (P = 0.049) were infected compared with adults, and no differences in prevalence were noted by sex, season of capture, or land use (rural vs. urban); however, significantly (P = 0.0370) higher worm burdens were found in infected raccoons from urban/suburban locations compared with rural areas. In addition, Toxascaris leonina , a morphologically similar ascarid, was found in 3 raccoons from Clarke County (n = 2) and Morgan County (n = 1). A second objective was to determine if sequence polymorphisms were associated with B. procyonis from different geographic regions. Because sequences from a single worm from Japan had been entered into GenBank, we obtained nematodes from Kentucky and Texas for comparison with our samples from Georgia. Sequence analysis of the 18S and 5.8S rRNA genes and the internal transcribed spacer (ITS) -1 and ITS-2 regions confirmed Georgia samples were B. procyonis. Although several polymorphic bases were observed within both ITS regions, none was associated with a particular geographic location. These data indicate that the distribution of B. procyonis within Georgia is increasing and only limited genetic variation is present in the rRNA and ITS gene regions among B. procyonis from the southern United States and introduced populations in Japan.     

Production and characterization of a monoclonal antibody for Pefloxacin and mechanism study of antibody recognition

In this report, an artificial antigen (PFLX–BSA: Pefloxacin connected bovine serum albumin) was successfully prepared. The monoclonal antibody against pefloxacin was produced and characterized using a direct competitive ELISA. The linear range of detection was 0.115–6.564 µg/L. The limit of detection defined as IC₁₅ was 0.170 ± 0.05 µg/L and the IC₅₀ was 0.902 ± 0.03 µg/L. The antibody variable region genes were amplified, assembled, and sequenced. A three–dimensional structural model of the variable region was constructed to study the mechanism of antibody recognition using molecular docking analysis. Three predicted essential amino acids, Thr53, Arg97 of heavy chain and Thr52 of light chain, were mutated to verify the theoretical model. Three mutants lost binding activity signi?cantly against pefloxacin as predicted. These may provide useful insights for studying antigen–antibody interaction mechanisms to improve antibody affinity maturation in vitro.     

Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies.

Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.     

Frequency of deposition and location of Baylisascaris procyonis eggs in raccoon feces

Baylisascaris procyonis is a large ascarid nematode found in the small intestine of raccoons (Procyon lotor). Infection with larvae of B. procyonis can produce visceral, ocular, and neural larval migrans in humans. Infected raccoons can shed millions of eggs a day in their feces. However, it is unknown whether eggs are consistently shed or whether eggs occur at irregular intervals by the population of female nematodes within a host. We trapped, infected, and collected daily fecal samples from 11 raccoons maintained in captivity. Eggs from B. procyonis were obtained from anterior, central, and posterior sections of raccoon feces, isolated by flotation, and quantified under 100× magnification. Naturally infected raccoons were collected and used as a comparison with the experimentally infected group. All raccoons in the experimental group (n=11) became infected with B. procyonis after consuming one infected mouse. Additionally, differential egg deposition rates were observed among individual raccoons from the experimental and naturally infected groups. Mean number of eggs per gram of feces (means±SE) was 16,563±4,321, which was less than previously reported for the species. However, no differences (F(2,30)=0.84, P=0.45) were noted in mean number of eggs per gram of feces among fecal sections. Wildlife biologists, veterinarians, health officials, and researchers of B. procyonis should collect daily fecal samples for a minimum of 3 days before identifying a raccoon as negative for B. procyonis infection. However, it does not matter where within the fecal matter the sample is obtained.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

First outbreak of Baylisascaris procyonis larva migrans in rabbits in Japan

A syndrome of progressive neurological signs was noticed in rabbits (Oryctolagus cuniculus) kept in a small wildlife park in mid-July 2000. Three out of 12 common raccoons (Procyon lotor) kept in this park were infected with Baylisascaris procyonis, and the larvae were found from affected rabbits. This outbreak is the first proven B. procyonis larva migrans in Japan, and the potential risk of serious zoonosis by this ascarid species should be considered by pet owners, veterinarians, physicians and public health authorities in this country as in North America where raccoons are endemic.     

Scanning electron microscopic observations of adult Baylisascaris procyonis (Nematoda)

The scanning electron microscope was used to illustrate the microtopographic features of the caudal end of adult male Baylisascaris procyonis. The male tail was relatively long, smoothly attenuated and often had a small button-like or mucronate termination. The preanal papillae were situated ventrally in two slightly divergent and somewhat irregularly spaced rows. Anterior and posterior to the anus were two slightly raised roughened patches consisting of several rows of small spines. Just anterior to the anus along the outer margin of the preanal roughened patch was a large double medioventral papilla. There were five pairs of postanal papillae with the first pair just posterior to the anus being double while the remaining four pairs were more closely associated in a group near the tail end. The second pair were also double papillae; however, in a few specimens they were not fused and appeared as two single closely associated papillae. The last three pairs of papillae were single. The fourth pair of caudal papillae were the phasmids and in the center of each was a ringed pore-like opening. The spicules of the male had a highly sculptured surface with a pincher-like terminal end.     

Land-use effects on prevalence of raccoon roundworm (Baylisascaris procyonis)

The raccoon (Procyon lotor) is the definitive host of Baylisascaris procyonis, a large intestinal roundworm that is zoonotic and can result in fatal or severe central nervous system disease in young children. Prevalence of infection among raccoon populations often is high, and in the midwestern United States, B. procyonis has been reported in 68-82% of raccoons. Raccoon populations have increased in response to changes in human land use, and often reach higher densities in urban and suburban landscapes than rural landscapes. However, shifts in foraging behavior among urban raccoons could impact the transmission of B. procyonis if small vertebrate intermediate hosts are not a significant part of the raccoon diet. The objective of this study was to compare prevalence of B. procyonis infection between urban and rural raccoon populations on a regional scale. Necropsy was done on 204 raccoons collected from September through February during 2000-2005 from seven states across the Midwest (regional sample). Baylisascaris procyonis was found in 54% of examined raccoons. Prevalence differed between land-use types (chi2=11.56, df=1, P=0.0007), and was higher among animals collected from rural locations (65%) than those collected in urban locations (41%). Intensity of infection also differed (F=5.52, df=1, P=0.02), with rural raccoons having greater worm burdens (x=29.63+/-36.42) than urban raccoons (x=13.85+/-18.47). Despite high densities of raccoons in urban landscapes, fewer urban raccoons were infected with B. procyonis, suggesting decreased dependence on intermediate hosts as a food source. This possible explanation was supported by a similar trend in prevalence among subsamples of raccoons collected from three Chicago-area populations (local samples) with differing levels of urbanization, population densities, and foraging behavior that had been intensively monitored during 1995-2002. Decreased transmission of B. procyonis in urban landscapes may be due to decreased predation of intermediate hosts, and contact of juvenile raccoons with B. procyonis eggs may be an important factor in maintaining infections within such populations.     

Generation and characterization of polyclonal and monoclonal antibodies to human NTPDase2 including a blocking antibody

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) is an ectonucleotidase that modulates P2 receptor activation by hydrolyzing ATP to ADP. In rodents, NTPDase2 is expressed by several specialized cell types such as vascular adventitial cells, neuroglial cells, hepatic portal fibroblasts, gustatory type I cells, and cells within the connective tissues of reproductive and gastrointestinal organs. Much less is known regarding the expression and function of NTPDase2 in humans. Here, we developed specific research tools to study human NTPDase2. We generated mouse monoclonal antibodies and rabbit polyclonal antibodies specific to human NTPDase2 and validated their specificity by western blot, immunocytochemistry, immunohistochemistry, and flow cytometry. In addition, one monoclonal antibody named hN2-D5 _s specifically inhibits human NTPDase2 enzymatic activity but not mouse nor rat NTPDase2. Using these antibodies, NTPDase2 immunoreactivity was detected on glial cells of the human enteric nervous system suggesting a function of the enzyme in intestinal motility. In conclusion, the new antibodies described in our work are novel tools that will enhance future studies of NTPDase2 expression and function in humans.     

Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae

When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.     

Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis

Two monoclonal antibodiesWuchereria bancrofti E 33 andWuchereria bancrofli E 34 raised againstWuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility.Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. WhenWuchereria bancrofti E 34 monoclonal antibody was used alongwith immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay. 68% of microfilaraemic sera (26 out of 38). 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected byWuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.     

Scanning electron microscopy of the labia of Baylisascaris procyonis (Nematoda)

The labial organization of adult Baylisascaris procyonis was studied by scanning electron microscopy, and found to be similar for males and females. The apical part of each lip was smooth, and the basal part reticulated. The dorsal lip possessed two dorsolateral double papillae and two internal labial papillae; the two subventral lips each had one ventrolateral double and one externolateral papilla, two internal labial papillae, and an amphid. The small papilla of each double set was dome-shaped and smooth, whereas the large papilla was broad and had a prominent central pore. The externolateral papillae had raised, highly sculptured surfaces with numerous slits and creases present. Amphids resembled those of previously studied nematodes. The internal labial papillae consisted of pits. Denticles arose as a single row from the apical edge of the inner labial surface, were usually evenly spaced, and pyramidal or conoidal in shape. They were typically unicuspid, but bicuspid denticles were occasionally seen. Denticle shape and size varied between specimens and on each specimen. A pit was seen in the cuticle in the central region of the denticular row of all lips. Several of these findings represent new information concerning ascaridoid nematodes.     

Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins.

Antibody response in mice to scrapie-associated fibril proteins (protease-resistant proteins [PrPs]) was generated to different epitopes depending on the source of antigen. Mice responded differently to PrPs isolated from scrapie-infected animals of homologous (mouse) versus heterologous (hamster) species. An enzyme-linked immunosorbent assay established to monitor this antibody response in mice immunized with PrPs was unable to detect such a response in scrapie-infected mice. A monoclonal antibody (MAb), 263K 3F4, derived from a mouse immunized with hamster 263K PrPs reacted with hamster but not mouse PrPs. MAb 263K 3F4 also recognized normal host protein of 33 to 35 kilodaltons in brain tissue from hamsters and humans but not from bovine, mouse, rat, sheep, or rabbit brains. This is the first demonstration of epitope differences on this host protein in different species. The defining of various epitopes on PrP through the use of MAbs will lead to a better understanding of the relationship of PrPs to their host precursor protein and to the infectious scrapie agent.     

Affinity purification of tetanus toxin using polyclonal and monoclonal antibody immunoadsorbents

Tetanus toxin has been immunopurified on immunoadsorbent columns derived from equine polyclonal antitoxin coupled to cyanogen bromide-activated Sepharose CL4B. Desorption of bound toxin in active form was achieved only when the immunoadsorbent was mixed with Sephadex G15 and this mixture overlaid on a further volume of Sephadex G15. With equine antibody, 64% of adsorbed toxin was recovered with a specific activity of 2400 limiting flocculation units (Lf)/mg protein N (1.2 × 10⁸ minimum lethal doses (MLD)/mg protein N). Similarly prepared immunoadsorbent derived from murine monoclonal antitoxin of low affinity had improved desorption with less acidic desorbents, without the requirement for Sephadex G15; greater than 80% of adsorbed toxin was recovered with a specific activity of 3000 Lf/mg protein N (1.6 × 10⁸ MLD/mg protein N).     

Affinity purification of tetanus toxin using polyclonal and monoclonal antibody immunoadsorbents

Tetanus toxin has been immunopurified on immunoadsorbent columns derived from equine polyclonal antitoxin coupled to cyanogen bromide-activated Sepharose CL4B. Desorption of bound toxin in active form was achieved only when the immunoadsorbent was mixed with Sephadex G15 and this mixture overlaid on a further volume of Sephadex G15. With equine antibody, 64% of adsorbed toxin was recovered with a specific activity of 2400 limiting flocculation units (Lf)/mg protein N (1.2 X 10(8) minimum lethal doses (MLD)/mg protein N). Similarly prepared immunoadsorbent derived from murine monoclonal antitoxin of low affinity had improved desorption with less acidic desorbents, without the requirement for Sephadex G15; greater than 80% of adsorbed toxin was recovered with a specific activity of 3000 Lf/mg protein N (1.6 X 10(8) MLD/mg protein N).     

Detection of DNA from the zoonotic raccoon roundworm Baylisascaris procyonis in a French wolf

Baylisascaris procyonis is a zoonotic nematode whose main definitive host is the raccoon, an invasive carnivore in Europe introduced from the United States. B. procyonis causes larva migrans with poor prognosis in humans. This parasite was unexpectedly detected in France for the first time upon molecular screening of wolf faecal samples. Because no patent infection was found, the wolf cannot be considered as a definitive host. This discovery of B. procyonis in France nonetheless raises questions about the parasite status of the expanding raccoon populations in the country, which will be investigated in the future.     

Survey of raccoons on Key Largo, Florida, USA, for Baylisascaris procyonis

Numbers of the endangered Key Largo woodrat (KLWR; Neotoma floridana smalli) have been declining for at least 25 yr. The raccoon (Procyon lotor) roundworm, Baylisascaris procyonis, has been found to have an adverse effect on the survival of Alleghany woodrats (N. magister). High densities of raccoons can exacerbate this problem by increasing the amount of feces containing viable eggs of B. procyonis available to woodrats. In 2002, 64 fecal samples were collected and examined for eggs of B. procyonis from >32 raccoons within the KLWR's known range on Key Largo, Florida, USA. All samples were negative for eggs of B. procyonis. Raccoon density in this area was approximately 0.62 raccoons/ha. Despite this high density of raccoons, B. procyonis does not appear to be a threat to the KLWR population.