Epicutaneous sensitization with nematode antigens of fish parasites results in the production of specific IgG and IgE.

Fish consumption plays an important role in the human diet. Hoplias malabaricus, trahira, is a freshwater fish widely appreciated in several Brazilian states and it is frequently infected by Contracaecum multipapillatum third-instar larvae (L3). The aim of the present study was to evaluate the allergenic potential of the C. multipapillatum L3 crude extract (CECM). BALB/c mice were immunized intraperitoneally (ip) with 10 or 50 µg CECM associated with 2 mg of aluminium hydroxide on days 0, 14 and 48. The determination of specific IgG and IgE antibody levels was done after immunization, and the late immunity was evaluated by the intradermal reaction in the ear pavilion. Epicutaneous sensitization was performed in the dorsal region, with antigenic exposure via a Finn-type chamber, containing 100 µg of chicken ovum albumin (OVA) or 100 µg CECM. After the exposures, the specific antibody levels were determined. In the ip immunization, there was a gradual increase in IgG antibody levels, independent of CECM concentration. In relation to IgE production, it was transitory, and immunization with 10 µg was more efficient than that of 50 µg. The same result was observed in the cellular hypersensitivity reaction. In the case of antigen exposure by the epicutaneous route, it was verified that only CECM was able to induce detectable levels of specific IgG and IgE antibodies. In the present study it was demonstrated that both intraperitoneal immunization and epicutaneous contact with C. multipapillatum larval antigens are potentially capable of inducing allergic sensitization in mice.

Influence of adult Anguillicoloides crassus load in European eels swimbladder on macrophage response.

Anguillicoloides crassus has become one of the most important threats to the European eel (Anguilla anguilla). Adult parasites colonize the swimbladder leading to an impaired functioning of this organ. The infection is also responsible for an increased in the stress level of infected eels, that could produce an altered immune response as well. Differences in parasite loads and effects in the European and Japanese eel (Anguilla japonica) have been described. We have studied the influence of the number of adult parasites present in the swimbladder of wild eels on the macrophage response (phagocytosis and respiratory burst) as part of the first immune response to pathogens. Our results show an increased phagocytozed bacterial survival 24 h post-infection in macrophages of eels infected with more than ten adult parasites compared to macrophages from eels infected with less than those ten adult parasites. Respiratory burst results also showed a less efficient response in macrophages from eels infected with more than ten adult parasites, although in this case results were not found to be significant.

Spirocerca lupi draft genome, vaccine and anthelmintic targets.

Spirocerca lupi is a parasitic nematode affecting predominantly domestic dogs. It causes spirocercosis, a disease that is often fatal. The assembled draft genome of S. lupi consists of 13,627 predicted protein-coding genes and is approximately 150 Mb in length. Several known anthelmintic gene targets such as for ß-Tubulin, glutamate, and GABA receptors as well as known vaccine gene targets such as cysteine protease inhibitor and cytokines were identified in S. lupi by comparing orthologs of C. elegans anthelmintic gene targets as well as orthologs to known vaccine candidates. New anthelmintic targets were predicted through an inclusion-exclusion strategy and new vaccine targets were predicted through an immunoinformatics approach. New anthelminthic targets include DNA-directed RNA polymerases, chitin synthase, polymerases, and other enzymes. New vaccine targets include cuticle collagens. These gene targets provide a starting platform for new drug identification and vaccine design.

Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction.

Macrophages are innate immune cells with essential roles in the immune response during helminth infection. Particularly, the direction of macrophage polarization could contribute to pathogen trapping and killing as well as tissue repair and the resolution of type 2 inflammation. This study establishes that the recombinant protein of Thelazia callipaeda macrophage migration inhibitory factor (T.cp-MIF) induces THP-1-derived macrophages to undergo M1 to M2 type dynamic polarization, using the methods of flow cytometry, real-time quantitative PCR, differential transcriptomic analysis and western blot. Interestingly, there was an increase in protein and mRNA expression of M1-type proteins and cytokines after the use of PI3K inhibitors, suggesting that the polarization state tends to favor the M1 type after M2 type inhibition. In conclusion, the dynamic polarization mechanism of T.cp-MIF-induced human THP-1-derived macrophages from M1 to M2 type is related to the binding of TLR4. It can first affect the M1 type polarization of macrophages by activating its downstream NF-κB pathway. Activation of the PI3K/Akt pathway and inhibition of NF-κB phosphorylation affects the M2 type polarization of macrophages.

Immunohistochemical characterization of lymphocyte and myeloid cell infiltrates in spirocercosis-induced oesophageal nodules.

Spirocerca lupi is a nematode that infects the dog's oesophagus and promotes the formation of an inflammatory fibroblastic nodule that progresses to sarcoma in approximately 25% of cases. Spirocercosis-associated oesophageal sarcoma is an excellent and under-utilized spontaneous model of parasite-associated malignancy. The inflammatory infiltrate of paraffin-embedded, non-neoplastic oesophageal nodules (n = 46), neoplastic nodules (n = 25) and normal oesophagus (n = 14) was examined by immunohistochemistry using MAC387 (myeloid cells), CD3 (T cells), Pax5 (B cells) and FoxP3 (T regulatory cells) antibodies. Myeloid cells predominated in 70% of nodules, in pockets around the worms' migratory tracts and in necro-ulcerative areas in neoplastic cases. T cells predominated in 23% of cases with a focal or diffuse distribution, in the nodule periphery. No significant differences were observed between neoplastic and non-neoplastic stages. FoxP3+ cells were observed in low numbers, not significantly different from the controls. The inflammation in spirocercosis is characterized by pockets of pus surrounded by organized lymphoid foci. There was no evidence of a local accumulation of FoxP3+ cells, unlike many previous studies that have reported an increase in FoxP3+ T cells in both malignancies and parasite infections. The triggering factor(s) driving the malignant transformation of the spirocercosis-associated chronic inflammatory nodule warrants further investigation.

Experimental infection with Anguillicola crassus alters immune gene expression in both spleen and head kidney of the European eel (Anguilla anguilla).

Invasive parasites have been implicated in the declines of several freshwater species. The swim bladder nematode Anguillicola crassus was introduced into Europe in the 1980s and is considered a threat to the European eel (Anguilla anguilla). Infection affects stress resistance and swimming behaviour. European eels produce an immune response against the parasite during the late stages of infection and after repeated infections. We used RNA-seq to examine the molecular response to infection during the poorly understood early stage and identify expression of genes and associated processes that are modified in two immune organs of European eels 3 days post infection with A. crassus. In the spleen, 67 genes were differentially expressed, 32 of which were annotated. Most of these were involved in immune processes and their regulation. Other differentially expressed genes in the spleen were important for heme metabolism and heme turn-over. In the head kidney, 257 genes (134 annotated) were differentially expressed. Several of these were associated with immune functions. Other differentially expressed genes in the head kidney were related to renal function, in particular osmoregulation and paracellular flow. We conclude that the early response of European eels to A. crassus is complex and involves various processes aside from the immune system. We identified molecular changes occurring early during the infection and identified candidate genes and processes which will facilitate future studies aimed at determining the factors affecting European eel viability in the face of this invasive parasite.

Co-expulsion of Ascaridia galli and Heterakis gallinarum by chickens.

Worm expulsion is known to occur in mammalian hosts exposed to mono-species helminth infections, whilst this phenomenon is poorly described in avian hosts. Mono-species infections, however, are rather rare under natural circumstances. Therefore, we quantified the extent and duration of worm expulsion by chickens experimentally infected with both Ascaridia galli and Heterakis gallinarum, and investigated the accompanying humoral and cell-mediated host immune responses in association with population dynamics of the worms. Results demonstrated the strong co-expulsion of the two ascarid species in three phases. The expulsion patterns were characterized by non-linear alterations separated by species-specific time thresholds. Ascaridia galli burden decreased at a daily expulsion rate (e) of 4.3 worms up to a threshold of 30.5 days p.i., followed by a much lower second expulsion rate (e = 0.46), which resulted in almost, but not entirely, complete expulsion. Heterakis gallinarum was able to induce reinfection within the experimental period (9 weeks). First generation H. gallinarum worms were expelled at a daily rate of e = 0.8 worms until 36.4 days p.i., and thereafter almost no expulsion occurred. Data on both humoral and tissue-specific cellular immune responses collectively indicated that antibody production in chickens with multispecies ascarid infections is triggered by Th2 polarisation. Local Th2 immune responses and mucin-regulating genes are associated with the regulation of worm expulsion. In conclusion, the chicken host is able to eliminate the vast majority of both A. galli and H. gallinarum in three distinct phases. Worm expulsion was strongly associated with the developmental stages of the worms, where the elimination of juvenile stages was specifically targeted. A very small percentage of worms was nevertheless able to survive, reach maturity and induce reinfection if given sufficient time to complete their life cycle. Both humoral and local immune responses were associated with worm expulsion.

Isolated optic neuritis from an identified Gnathostoma spinigerum.

PURPOSE: To describe a patient with isolated monocular optic neuritis caused by an identified Gnathostoma spinigerum infestation. CASE REPORT: A 21-year-old man developed a swollen eyelid and painful monocular visual loss of his left eye which did not improve after treatment by intravenous steroid and albendazole. A remarkable eosinophilia in his peripheral blood count was demonstrated. The patient subsequently found a live parasite emerged from his lower eyelid and it was successfully removed by himself. Gross and histopathology examinations of the obtained parasite was undertaken. The parasite was identified as Gnathostoma spinigerum. His blood test for Gnathostoma antibody was positive. DISCUSSION: The etiology of isolated optic neuritis in this patient was Gnathostoma spinigerum which was confirmed by the histopathology of the obtained parasite and the positive serologic test. CONCLUSIONS: We could identify the exact parasite that was proven to cause an isolated optic neuritis. The immediate removal of a causative parasite maynot result in an improvement of the injured tissue but is beneficial in preventing further destruction as well as future complications.

Differential Evolutionary Selection and Natural Evolvability Observed in ALT Proteins of Human Filarial Parasites.

The abundant larval transcript (ALT-2) protein is present in all members of the Filarioidea, and has been reported as a potential candidate antigen for a subunit vaccine against lymphatic filariasis. To assess the potential for vaccine escape or heterologous protection, we examined the evolutionary selection acting on ALT-2. The ratios of nonsynonymous (K(a)) to synonymous (K(s)) mutation frequencies (ω) were calculated for the alt-2 genes of the lymphatic filariasis agents Brugia malayi and Wuchereria bancrofti and the agents of river blindness and African eyeworm disease Onchocerca volvulus and Loa loa. Two distinct Bayesian models of sequence evolution showed that ALT-2 of W. bancrofti and L. loa were under significant (P<0.05; P < 0.001) diversifying selection, while ALT-2 of B. malayi and O. volvulus were under neutral to stabilizing selection. Diversifying selection as measured by ω values was notably strongest on the region of ALT-2 encoding the signal peptide of L. loa and was elevated in the variable acidic domain of L. loa and W. bancrofti. Phylogenetic analysis indicated that the ALT-2 consensus sequences formed three clades: the first consisting of B. malayi, the second consisting of W. bancrofti, and the third containing both O. volvulus and L. loa. ALT-2 selection was therefore not predictable by phylogeny or pathology, as the two species parasitizing the eye were selected differently, as were the two species parasitizing the lymphatic system. The most immunogenic regions of L. loa and W. bancrofti ALT-2 sequence as modeled by antigenicity prediction analysis did not correspond with elevated levels of diversifying selection, and were not selected differently than predicted antigenic epitopes in B. malayi and O. volvulus. Measurements of ALT-2 evolvability made by χ2 analysis between alleles that were stable (O. volvulus and B. malayi) and those that were under diversifying selection (W. bancrofti and L. loa) indicated significant (P<0.01) deviations from a normal distribution for both W. bancrofti and L. loa. The relationship between evolvability and selection in L. loa followed a second order polynomial distribution (R2 = 0.89), indicating that the two factors relate to one another in accordance with an additional unknown factor. Taken together, these findings indicate discrete evolutionary drivers acting on ALT-2 of the four organisms examined, and the described variation has implications for design of novel vaccines and diagnostic reagents. Additionally, this represents the first mathematical description of evolvability in a naturally occurring setting.

A case report of colonic ileus due to eosinophilic nodular lesions caused by Gnathostoma doloresi infection.

Gnathostomiasis is primarily a disease of the skin characterized as creeping eruption or mobile erythema. However, larval Gnathostoma sometimes migrate into an unexpected site to elicit serious illness. Here we describe a case of colonic ileus caused by Gnathostoma doloresi. The patient was a 57-year-old man living in Miyazaki Prefecture, Japan, which is known as an area endemic for this parasite. One week after having eaten a few slices of the flesh of a snake (Agkistrodon halys), he developed severe abdominal pain. An abdominal radiograph revealed multiple gas-fluid levels with a distended bowel of an inverted U shape. A barium enema revealed a tumor in the ascending colon near the hepatic flexure that was surgically removed by simple colonic resection. An oblique section of a parasite surrounded by massive infiltration of eosinophils was found by postoperative histopathologic examination. The entire body of the advanced third-stage larva of G. doloresi was dissected from a specimen-embedded paraffin block.

Evaluation of an ELISA and immunoblotting for studying the humoral immune response in Anguillicola crassus infected European eel Anguilla anguilla.

The applicability of an enzyme-linked immunosorbent assay (ELISA) for the detection of anguillicolosis in feral eels was examined using a crude antigen preparation from the body wall of adult Anguillicola crassus. The screening consisted of samples from 100 feral European eels Anguilla anguilla. As a reference the actual status of infection was determined by dissection of the eels' swim-bladders. The ELISA results were compared with a background value calculated from the results obtained from 43 non-infected farm eels. The screened samples had a high prevalence of A. crassus (83 %); however, the specificity and the negative predictive value of the ELISA were low compared to the high positive predictive value. Nonetheless, the reproducibility (precision) of the test was satisfactory, and for the non-infected reference group specificity was 97.7 %. Although the ELISA, as used in the present study, is not applicable for diagnostic purposes, it represents a useful tool for the investigation of the specific humoral immune response of eels against A. crassus under controlled experimental conditions. Immunoblots using crude antigen preparations from different parts of adult A. crassus as well as a crude somatic third-stage (L3) antigen preparation illustrated that only antigens associated with the body wall of adult A. crassus are potentially suitable for diagnostic purposes. Despite the fact that antibodies against Raphidascaris acus cross-reacted with 3 body wall antigens of A. crassus, the most encouraging results were obtained with the antigen preparation from the outer cuticle of adult A. crassus which yielded a conspicuous, broad band at about 100 kDa.

Characterization of monoclonal antibodies against Gnathostoma nipponicum.

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.

Third-stage larvae of Daniconema anguillae (Nematoda: Dracunculoidea) in the subcutaneous tissue of eel Anguilla anguilla.

Daniconema anguillae Moravec et Køie, 1987 larvae measuring 1.64-1.76 mm were occasionally found in considerable numbers in the fins and subcutaneous connective tissue of approximately 50% of eel Anguilla anguilla (L.) sampled from Lake Balaton, Hungary. The larvae were noted for their slender body, very long tail with a rounded tip, a densely transversely striated cuticle, and the presence of boring tooth and large kidney-shaped amphids on the cephalic end. The larvae could easily be recovered from the above mentioned organs by placing them into isotonic saline solution. No disease signs or pathological changes attributable to the larval infection could be observed. The only histological indication of host reaction was the appearance of macrophages adhering to the body surface of larvae and of cells with spherical nucleus in areas around the larvae. A possible life cycle pattern of D. anguillae is discussed.

Host tissue reaction to Gnathostoma malaysiae (Nematoda: Gnathostomidae) in Rattus surifer Miller.

The occurrence of adult Gnathostoma malaysiae in Rattus surifer and R. tiomanicus in Malaysia has been reported but there are no known reports on the host tissue reactions. This paper reports on the gross pathology caused by G. malaysiae in a red spiny forest rat, R. surifer and the tissue reactions caused. A tumor-like growth was located on the mid-stomach wall in a female rat captured in Gunung Bachock, Kelantan, Malaysia. This growth consisted of four tunnel-like structures containing sanguinopurulent fluid and leukocytes and this structure led into a central canal. The tissue surrounding the tumor was greatly inflamed and there was localized gastritis. The tunnel-like structure was surrounded by dense fibrotic tissue. The stomach wall was devoid of superficial epithelium and smooth muscle but mucinous glands were present. The midregion of the fibrotic scar contained eggs of G. malaysiae which had evoked a strong tissue reaction and were surrounded by pus. Blood vessels were empty, dilated and had undergone vasculitis and thrombosis.

Immunohistochemical localization of Gnathostoma spinigerum larval antigens by monoclonal antibodies: I. Light microscopy.

Immunohistochemical localization of antigens in advanced third-stage larvae of Gnathostoma spinigerum (GsAL3) was studied by indirect enzyme immunostaining using 7 G. spinigerum specific monoclonal antibodies, FS-3D11, SS-5H5, SK-6C4, SK-4E1, SK-7G6, SD-8D4 and SA-9B5. All these MAb belong to the IgG1 subclass and only FS-3D11 and SS-5H5 recognize carbohydrate determinants. Each MAb exhibited a different reaction pattern and staining intensity in sectioned GsAL3. FS-3D11 bound primarily to the intestinal brush border whereas SS-5H5 reacted with various tissues of the parasite including intestinal epithelium and brush border, lateral cords, muscle, pseudocoel, and cuticle. SK-6C4 predominantly stained muscle, however, SK-4E1 and SK-7G6 exhibited a lack of labeling. SD-8D4 bound to the cuticle and the lateral cords whereas SA-9B5 reacted primarily with the pseudocoel. These results suggest that antigens sharing common epitopes are present in various structures of the larvae with the intestine being the most antigenic site. The present data also suggest that certain GsAL3 antigens recognized by the MAb obtained in this study are sensitive to formalin fixation and/or paraffin embedding since for 2 out of the 7 MAb staining was negative.

Antigenic components of Gnathostoma spinigerum recognized by infected human sera by two-dimensional polyacrylamide gel electrophoresis and immunoblotting.

Antigenic components of Gnathostoma spinigerum larval extract were revealed by two-dimensional gel electrophoresis (2-DE) and immunoblot analysis using sera from patients with 6 proven cases of gnathostomiasis, 5 presumptive cases of gnathostomiasis, 3 proven cases of angiostrongyliasis, 3 proven cases of cysticercosis, and pooled sera from healthy adults. By the 2-DE, the larval extract was highly complex and consisted of more than 75 polypeptides. Immunoblotting analysis of this larval extract after reaction with each of 6 proven gnathostomiasis sera revealed various numbers of antigenic spots ranging from 30 to 70 spots at the approximate molecular masses of less than 14.4 to more than 94 kDa with isoelectric points (pI) of less than 4.65 to 9.6. Antigenic spots at the approximate molecular mass of more than 30 kDa were recognized with the proven angiostrongyliasis, proven cysticercosis and healthy control sera but these sera did not react with the spots at approximate molecular masses of 23-25 kDa with pI of 8.3-8.5. The reacted spots, which consisted of at least 1 to 2 spots, were unique for the recognition of gnathostomiasis sera. Five out of 6 (83.3%) proven and 4 out of 5 (80%) presumptive gnathostomiasis sera reacted with these specific spots.

Attempts to analyse Anguillicola crassus infection and the humoral host response in eels (Anguilla anguilla) of Lake Balaton, Hungary.

Since the introduction of Anguillicola crassus into Europe, anguillicolosis has been a considerable problem in several countries. From 1991, periodical eel mortality occurred in Lake Balaton, Hungary. However, eels with a worm burden of 20 to 50 parasites did not show severe swimbladder lesions, which observation cast doubts on the primary aetiological role of the parasite in the eel kill. In order to study the pathology of the infection, from the spring of 1996 until October of the same year, 51 eels were collected from two regions of Lake Balaton and examined for swimbladder changes. To detect humoral antibodies, an enzyme-linked immunosorbent assay (ELISA) was performed, using cuticular-oesophageal worm antigen. The results of the test show the applicability of the method. However, no direct correlation was found between antibody levels or the intensity of infection and the swimbladder lesions. The low level of specific antibodies and the increasing severity of swimbladder changes in the autumn suggest that parasite-induced immunity is insufficient to prevent reinfection.

Specific IgE antibody responses to somatic and excretory-secretory antigens of third stage G. spinigerum larvae in human gnathostomiasis.

Specific IgE antibody levels in the serum of patients with proven gnathostomiasis and in those with intermittent cutaneous migratory swelling (CMS) were determined by the enzyme-linked immunosorbent assay (ELISA) using somatic extract and excretory-secretory (ES) products of Gnathostoma spinigerum infective larvae as antigens. The third stage larval used were obtained from naturally infected eels. There was an increase in specific IgE antibody to both antigens in these patients. The mean levels of these specific IgE antibodies were significantly higher than that of the healthy control (P<0.01). Comparison between using somatic extract and ES products in the test showed, a positive result in the group of suspected patients with gnathostomiasis or CMS was significantly higher when using ES products (81.81%) than somatic extract (59.09%) as the antigens (P<0.05). However, both somatic and ES antigens cross-reacted with other parasitic sera. The overall sensitivity of the ELISA for these IgE antibodies detection were 71.87 per cent and 87.50 per cent with somatic and ES antigens, respectively. The specificity was 57.53 per cent when somatic antigen was used and increased to 69.86 per cent when ES antigen was used. The positive and negative predictive values of the test were 42.59 per cent and 82.35 per cent by using somatic antigen. Both of these values, were also increased to 56.00 per cent and 92.72 per cent by using the ES antigen. It is obvious that more potential components may be present in ES products than those in the somatic extract. The ES antigen may have to be further purified and may be suitable for evaluation of the effectiveness of chemotherapy. As such, the antibody responses to secreted products are more closely related to active infection than the anti-whole worm antibody that may persist following the death of the parasites. However, in this disease, the effect of the IgE antibody on its pathophysiology it is still not known.

Gnathostomiasis possibly caused by Gnathostoma malaysiae.

Gnathostomiasis is rarely reported in travelers, although the disease remains a major public health problem in Southeast Asia. A creeping eruption and Quincke's edema (slowly migrating erythema with pruritus) appeared in two Japanese men who had eaten raw freshwater shrimp in Myanmar. A Gnathostoma larva was found in subcutaneous tissue from one of the men. Four species causing human gnathostomiasis, G. hispidum, G. doloresi, G. nipponicum and G. spinigerum, can be distinguished based on the number of nuclei in intestinal epithelial cells of infected larvae, in cross-section. In G. hispidum, only a single large nucleus is found. Morphologically, our larva was initially identified as G. hispidum. However, since the number of epithelial cells was greater and the body width was larger than those of a "large-type" 3rd-stage larva of G. hispidum, the larva was then identified as a 3rd-stage larva of G. malaysiae, Miyazaki and Dun, 1965, as reported by Setasuban et al, (1991). Since no human cases caused by this species of Gnathostoma have previously been encountered, this appears to be the first report of gnathostomiasis due to G. malaysiae.