Antigen detection: Insights into Toxocara and other ascarid infections in dogs and cats.

Detection of ascarid excreted or secreted (E/S) molecules is an alternative approach to the identification of infection by egg flotation. E/S molecules serve as direct markers for the ascarid nematode commonly found in cats and dogs (Toxocara spp., Toxascaris leonina and Baylisascaris procyonis). The nematode derived E/S material mixes with the intestinal contents of the host animal and is available for detection as a coproantigen in the host's faeces. Antigen capture immunological techniques allow sensitive coproantigen detection. Different patterns of antigen to egg agreement are demonstrated in an experimental Toxocara canis infection throughout the prepatent, patent, and post-treatment phases. Examination of faecal samples from a large field population of dogs and cats tested for both egg shedding and antigen indicates that more infections were identified by antigen. Host age influences the agreement of antigen and Toxocara egg results. Older dogs and cats were less likely to have a patent infection (egg positive and antigen positive) result pattern. An egg observation in the absence of antigen detection may indicate a spurious egg. The impact of spurious eggs was further examined by comparisons of cohorts of dogs separated by presence or absence of a pseudoparasite observation or by egg semi-quantification bin. Lastly, the antigen to egg agreement was calculated for other ascarid species.

Local immune depression in Baltic cod (Gadus morhua) liver infected with Contracaecum osculatum.

Third-stage larvae of the anisakid nematode Contracaecum osculatum infecting cod (Gadus morhua) liver elicit a host immune response involving both innate and adaptive factors, but the reactions differ between liver and spleen. Inflammatory reactions occur in both liver and spleen, but a series of immune effector genes are downregulated in liver infected with nematodes whereas these genes in spleen from the same fish are upregulated. A series of novel primer and probe sets targeting cod immune responses were developed and applied in a real-time quantitative polymerase chain reaction set-up to measure the expression of immune-relevant genes in liver and spleen of infected and uninfected cod. In infected liver, 12 of 23 genes were regulated. Genes encoding cytokines associated with inflammatory reactions (IL-1ß, IL-6, IL-8) were significantly upregulated, whereas genes encoding effector molecules, assisting the elimination of pathogens, C-reactive protein (CRP)-PII, hepcidin, lysozyme G1, lysozyme G2, C3 and IgDm, were significantly downregulated. The number of downregulated genes increased with the parasite burden. In spleen, 14 of 23 immune genes showed significant regulation and nine of these were upregulated, including genes encoding CRPI, CRPII, C3, hepcidin and transferrin. The general gene expression level was higher in spleen compared to liver, and although inflammation was induced in nematode-infected liver, the effector molecule genes were depressed, which suggests a worm-induced immune suppression locally in the liver.

Resistance and tolerance to mixed nematode infections in chicken genotypes with extremely different growth rates.

Fast growing broilers are less able to cope with fitness related challenges. As the allocation of metabolic resources may be traded off between performance and defence functions in parasitized hosts, we hypothesized that fast growing broilers are more sensitive to mixed nematode infections compared with slower growing genotypes under the same environmental conditions. Therefore, we compared male birds of genotypes selected for either meat production (Ross-308, R) or egg production (Lohmann Brown Plus, LB) or for both purposes (Lohmann Dual, LD), to assess their resistance and tolerance to mixed nematode infections with Ascaridia galli and Heterakis gallinarum. While infections reduced feed intake in all three genotypes, feed conversion efficiency was not affected. Infections impaired growth performance only in R birds, indicating lower tolerance in the fast growing genotype compared with slower growing LB and LD genotypes. Impaired tolerance in R birds was associated with a relative nutrient scarcity due to an infection-induced lower feed intake. Resistance to experimentally induced infections depended on host genotype as well as on the worm species involved. Overall, the A. galli burden was higher in R than LB, whereas the burden of LD was not different from that of R and LB. In contrast, the H. gallinarum burden of first generation worms was similar in the three genotypes. Susceptibility to re-infection with H. gallinarum was higher in LB than in LD, whereas very low levels of re-infection were observed in R birds. Our data collectively suggest that resistance and tolerance to mixed nematode infections are sensitive to growth rate in chickens. These differences amongst genotypes may partly be associated with a mismatch between the actual nutrient supply and genotype-specific nutrient requirements.

Transcriptome and metabolome analyses of Coilia nasus in response to Anisakidae parasite infection.

Parasites from the family Anisakidae are capable of infecting a range of marine fish species worldwide. Coilia nasus, which usually feeds and overwinters in coastal waters and spawns in freshwater, is highly susceptible to infection by Anisakidae. In this study, we used scanning electron microscopes to show that C. nasus infected by Anisakidae exhibited damage and fibrosis of the liver tissue. To better understand host immune reaction and metabolic changes to Anisakidae infection, we used a combination of transcriptomic and metabolomic method to characterize the key genes and metabolites, and the signaling pathway regulation of C. nasus infected by Anisakidae. We generated 62,604 unigenes from liver tissue and identified 391 compounds from serum. Of these, Anisakidae infection resulted in significant up-regulation of 545 genes and 28 metabolites, and significant down-regulation of 416 genes and 37 metabolites. Seventy-four of the 961 differentially expressed genes were linked to immune response, and 1, 2-Diacylglycerol, an important immune-related metabolite, was significantly up-regulated after infection. Our results show activation of antigen processing and presentation, initiation of the T cell receptor signaling pathway, disruption of the TCA cycle, and changes to the amino acid and Glycerolipid metabolisms, which indicate perturbations to the host immune system and metabolism following infection. This is the first study describing the immune responses and metabolic changes in C. nasus to Anisakidae infection, and thus improves our understanding of the interaction mechanisms between C. nasus and Anisakidae. Our findings will be useful for future research on the population ecology of C. nasus.

Detection and Evaluation of Antibody Response to a Baylisascaris-Specific Antigen in Rodent Hosts with the Use of Western Blotting and Elisa.

Diagnosis of parasitic diseases that involve tissue-stage larvae is challenging, and serology remains the most effective antemortem test for detecting these infections. Baylisascaris procyonis, the raccoon roundworm, is a zoonotic ascarid. Raccoons are the usual definitive host, and humans may be infected as accidental hosts. More than 150 species of birds and mammals may act as paratenic hosts, and rodents play an important role in the transmission and maintenance of this parasite in nature. Migratory larvae in paratenic host tissues can produce ocular disease and severe to fatal neurologic disease, but not all infected hosts develop signs. A sensitive and specific Western blot (WB) assay based on a recombinant Baylisascaris-specific antigen (rBpRAG-1) has been developed for use in humans. We evaluated the use of this antigen to detect Baylisascaris spp. infections in rodent paratenic hosts. With the use of 4 species of Peromyscus mice ( Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, Peromyscus polionotus) from a previous infection trial, we developed species-adapted WB and ELISA assays and evaluated performance compared to detection of larvae in tissue samples. These assays revealed species-level differences in seroconversion and terminal antibody concentrations, with P. leucopus developing significantly greater antibody concentrations than P. californicus and P. polionotus at all dose levels, and P. maniculatus at the low dose. Some P. californicus and P. polionotus failed to seroconvert despite the recovery of larvae from their tissues. WB and ELISA results were correlated; however, the WB demonstrated higher sensitivity than the ELISA overall (72.2% versus 63.9%, respectively). With the use of experimental samples, specificity was 100% for WB and 94.1% for ELISA. A WB was also used to test Mus and Rattus samples, and although numbers were too limited to evaluate sensitivity and specificity, all animals known to be infected by tissue digestion were WB positive, and all uninfected animals were negative. Finally, the Peromyscus-adapted WB and ELISA were used to test a set of serum samples from wild-trapped P. maniculatus and Rattus rattus. Both assays were generally sensitive, but specificity was equivocal. This emphasizes the challenge of using serology for investigation of wildlife diseases, in which hosts have unknown exposure histories. Nevertheless, serologic methods have utility in the study of Baylisascaris spp. in paratenic hosts, either wild or captive, and have advantageous attributes (non-lethal, high-throughput), but results should be interpreted carefully.

Immunomodulatory effects of excretory/secretory compounds from Contracaecum osculatum larvae in a zebrafish inflammation model.

Excretory/secretory (ES) compounds isolated from third-stage larvae of the anisakid nematode Contracaecum osculatum parasitizing liver of Baltic cod were investigated for effects on immune gene expression in a zebrafish LPS-induced inflammation model. ES products containing a series of proteins, of which some had enzymatic activity, were injected solely or with LPS. ES proteins alone induced up-regulation of a number of immune-related genes, but generally to a lower degree compared to LPS. When co-injected with LPS, the worm products exacerbated merely expression of five genes affecting Th1, Th2, Th17 and innate responses compared to the LPS-injected group. However, the level of overexpression decreased in an inverse dose-dependent manner. The immune regulating action of C. osculatum ES products is interpreted as an important evolutionary ability of larval parasites in the transport host which makes it less susceptible to host immune responses whereby the probability of reaching the final host is increased.

Early-Life Diet Affects Host Microbiota and Later-Life Defenses Against Parasites in Frogs.

Food resources can affect the health of organisms by altering their symbiotic microbiota and affecting energy reserves for host defenses against parasites. Different diets can vary in their macronutrient content and therefore they might favor certain bacterial communities of the host and affect the development and maintenance of the immune system, such as the inflammatory or antibody responses. Thus, testing the effect of diet, especially for animals with wide diet breadths, on host-associated microbiota and defenses against parasites might be important in determining infection and disease risk. Here, we test whether the early-life diet of Cuban tree frogs (Osteopilus septentrionalis) affects early- and later-life microbiota as well as later-life defenses against skin-penetrating, gut worms (Aplectana hamatospicula). We fed tadpoles two ecologically common diets: a diet of conspecifics or a diet of algae (Arthrospira sp.). We then: (1) characterized the gut microbiota of tadpoles and adults; and (2) challenged adult frogs with parasitic worms and measured host resistance (including the antibody-mediated immune response) and tolerance of infections. Tadpole diet affected bacterial communities in the guts of tadpoles but did not have enduring effects on the bacterial communities of adults. In contrast, tadpole diet had enduring effects on host resistance and tolerance of infections in adult frogs. Frogs that were fed a conspecific-based diet as tadpoles were more resistant to worm penetration compared with frogs that were fed an alga-based diet as tadpoles, but less resistant to worm establishment, which may be related to their suppressed antibody response during worm establishment. Furthermore, frogs that were fed a conspecific-based diet as tadpoles were more tolerant to the effect of parasite abundance on host mass during worm establishment. Overall, our study demonstrates that the diet of Cuban tree frog tadpoles affects the gut microbiota and defenses against parasitic gut worms of frogs, but these effects depend on the stage of the host and infection, respectively.

Seroprevalence of Baylisascaris procyonis Infection among Humans, Santa Barbara County, California, USA, 2014-2016.

Baylisascaris procyonis (raccoon roundworm) infection is common in raccoons and can cause devastating pathology in other animals, including humans. Limited information is available on the frequency of asymptomatic human infection. We tested 150 adults from California, USA, for B. procyonis antibodies; 11 were seropositive, suggesting that subclinical infection does occur.

Nodular typhlocolitis, heterakiasis, and mesenchymal neoplasia in a ring-necked pheasant ( Phasianus colchicus) with immunohistochemical characterization of visceral metastases.

A 9-y-old, male ring-necked pheasant ( Phasianus colchicus) was autopsied following euthanasia because of acute distress, recumbency, and dyspnea. The bird had experienced a protracted period of neuromuscular disease localized to the left sciatic nerve. Gross and histologic examination of the large intestine revealed intramural nodules predominantly comprised of atypical, whorling spindle cells with small cores of granulomatous inflammation centered on cross-sections of immature adult nematodes. The body structures of these metazoan organisms and clinical disease manifestation are consistent with Heterakis isolonche infection. Nodular spindle cell proliferations without granulomatous inflammation or intralesional nematodes were also found throughout the liver and lungs, suggesting metastasis from the intestine. Immunohistochemical staining of the hepatic and pulmonary tumor tissue with vimentin and S100 suggests a neurofibroblastic origin.

Equine antibody response to larval Parascaris equorum excretory-secretory products.

Parascaris equorum is an intestinal nematode of foals and young horses that can produce mild to severe pathology. Current diagnosis is limited to detection of patent infections, when parasite eggs are identified during fecal examinations. This study examined the use of larval P. equorum excretory-secretory (ES) products in a western blot test for diagnosis of prepatent equine P. equorum infection. Sera from adult mares negative for patent P. equorum infections, foals prior to consuming colostrum, and P. equorum infected foals were used as controls in this study. Study samples included sera from 18 broodmares prior to parturition and sera from their foals throughout the process of natural infection. Sera from study horses were examined for IgG(T) antibody recognition of ES products. Foals naturally infected with P. equorum possessed IgG(T) antibodies against 19kDa, 22kDa, 26kDa, and 34kDa ES products. However, passive transfer of colostral antibodies from mares was shown to preclude the use of the crude larval ES product-based western blot test for diagnosis of prepatent P. equorum infections in foals.

Anguilla anguilla intestinal immune response to natural infection with Contracaecum rudolphii A larvae.

The European eel, Anguilla anguilla, is a major warm-water fish species cultured in North and South Europe. Seventy-one A. anguilla collected between 2010 and 2015 from the Comacchio lagoons were examined. Fish were infected and damaged by larvae (L3) of the nematode Contracaecum rudolphii A, which were encapsulated within the thickness of the intestinal wall and within the external visceral peritoneum (serosa). Conspicuous granulomas, visible at sites of infection, were arranged in a trilayer, formed by a series of concentric whorls. The cells involved in the immune response and their distribution in the granuloma layers were assessed by immunohistochemical, immunofluorescence, and ultrastructural techniques. The outer part of the granuloma contained macrophages, macrophage aggregates, and mast cells (MCs) scattered among fibroblasts. This layer was vascularized, with degranulation of MCs occurring in close proximity to the capillaries. The middle layer was rich in MCs and fibroblasts. The inner layer, closest to the parasite larva, consisted mainly of dark epithelioid cells, some of which were necrotic. Non-necrotic epithelioid cells formed desmosomes between themselves or with fibroblasts. Within the granulomas, numerous cells of different types were positive to proliferative cell nuclear antigen antibody, indicating a high degree of cellular proliferation around the larvae.

Potential of recombinant inorganic pyrophosphatase antigen as a new vaccine candidate against Baylisascaris schroederi in mice.

The intestinal nematode Baylisascaris schroederi is an important cause of death for wild and captive giant pandas. Inorganic pyrophosphatases (PPases) are critical for development and molting in nematode parasites and represent potential targets for vaccination. Here, a new PPase homologue, Bsc-PYP-1, from B. schroederi was identified and characterized, and its potential as a vaccine candidate was evaluated in a mouse challenge model. Sequence alignment of PPases from nematode parasites and other organisms show that Bsc-PYP-1 is a nematode-specific member of the family I soluble PPases. Immunohistochemistry revealed strong localization of native Bsc-PYP-1 to the body wall, gut epithelium, ovary and uterus of adult female worms. Additionally, Bsc-PYP-1 homologues were found in roundworms infecting humans (Ascaris lumbricoides), swine (Ascaris suum) and dogs (Toxocara canis). In two vaccine trials, recombinant Bsc-PYP-1 (rBsc-PYP-1) formulated with Freund complete adjuvant induced significantly high antigen-specific immunoglobulin (Ig)G but no IgE or IgM responses. Analysis of IgG-subclass profiles revealed a greater increase of IgG1 than IgG2a. Splenocytes from rBsc-PYP-1/FCA-immunized mice secreted low levels of T helper (Th)1-type cytokines, interferon-γ and interleukin (IL)-2, while producing significantly high levels of IL-10 and significantly elevated levels of IL-4 (Th2 cytokines) after stimulation with rBsc-PYP-1 in vitro. Finally, vaccinated mice had 69.02-71.15% reductions (in 2 experiments) in larval recovery 7 days post-challenge (dpc) and 80% survival at 80 dpc. These results suggest that Th2-mediated immunity elicited by rBsc-PYP-1 provides protection against B. schroederi, and the findings should contribute to further development of Bsc-PYP-1 as a candidate vaccine against baylisascariasis.

Galectins in the abdominal cavity of the conger eel Conger myriaster participate in the cellular encapsulation of parasitic nematodes by host cells.

Congerin is a proto-type galectin distributed on the skin and mucosal epithelia of the upper digestive tract of the Japanese conger eel Conger myriaster. It has at least 2 isotypes, namely, congerin I and II, and plays a role in bio-defense at the body surface. In the current study, we identified both isotypes in the peritoneal fluid and peritoneal cells of C. myriaster by western blot and mass spectrometry (MS)/MS analysis. Cucullanus nematodes parasitize the abdominal cavity of C. myriaster, and immunohistochemical analyses demonstrated that congerins can bind to both the body surface of the encapsulated nematodes and the encapsulating cells. Furthermore, adhesion of the peritoneal cells to Sepharose particles was greatly accelerated when the microspheres were coated with congerin. Indeed, this effect was significantly hampered by the addition of lactose. These results indicate that congerin participates in the cellular encapsulation of the Cucullanus nematode via the induction of cellular adhesion to the parasites depending on lectin-glycoside recognition.

Experimental lagochilascariosis in X-chromosome-linked immunodeficient mice.

Lagochilascaris minor is the etiological agent of lagochilascariosis, a disease that affects the neck region and causes exudative abscesses, with eggs, adult parasites and L3/L4 larvae in the purulent exudates. Mice are now considered to be intermediate hosts for the parasite. To determine the pattern of infection in B1 cell-deficient mice, experimental lagochilascariosis was studied in BALB/c and X-chromosome-linked immunodeficient (xid) mice. BALB.xid-infected mice showed lower numbers of larvae. Third-stage larvae, fourth-stage larvae and adult parasites were found in both strains. BALB/c mice produced IgM, IgG, IgA and IgE against the crude extract and secreted/excreted antigens of the parasite. On the other hand, BALB.xid mice did not produce IgM and produced lower levels of IgG and IgA, and similar quantities of IgE.

Experimental lagochilascariosis in X-chromosome-linked immunodeficient mice / Lagochilascariose experimental em camundongos com imunodeficiência ligada ao cromossomo X

Lagochilascaris minor is the etiological agent of lagochilascariosis, a disease that affects the neck region and causes exudative abscesses, with eggs, adult parasites and L3/L4 larvae in the purulent exudates. Mice are now considered to be intermediate hosts for the parasite. To determine the pattern of infection in B1 cell-deficient mice, experimental lagochilascariosis was studied in BALB/c and X-chromosome-linked immunodeficient (xid) mice. BALB.xid-infected mice showed lower numbers of larvae. Third-stage larvae, fourth-stage larvae and adult parasites were found in both strains. BALB/c mice produced IgM, IgG, IgA and IgE against the crude extract and secreted/excreted antigens of the parasite. On the other hand, BALB.xid mice did not produce IgM and produced lower levels of IgG and IgA, and similar quantities of IgE.

Lagochilascaris minor é o agente etiológico da lagochilascariose, uma doença que afeta a região de pescoço provocando abscessos exudativos contendo ovos, parasitas adultos e larvas L3/L4 nos exudates purulentos. Atualmente, camundongos são considerados hospedeiros intermediários do parasita. Para determinar o padrão de infecção em camundongos deficientes de células B1, a lagochilascariose experimental foi estudada em camundongos BALB/c e em camundongos com imunodeficiência ligada ao cromossomo X (xid). Camundongos BALB.xid infectados mostraram menor número de larvas. Larvas L3, L4 e parasitas adultos foram encontrados em ambas as linhagens. Camundongos BALB/c produziram IgM, IgG, IgA e IgE contra o extrato bruto e antígenos secretados/excretados do parasita; por outro lado, camundongos BALB.xid não produziram IgM, produziram baixos níveis de IgG e IgA, e quantidades semelhantes de IgE.

Cloning, expression and evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag3 antigen in mice.

The gene Bs-Ag3 enconding a antigen of 37kDa from Baylisascaris schroederi (giant panda isolates), as well as the recombinant Bs-Ag3, obtained by cloning and expression of the Bs-Ag3 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in BALB/c mice against L3-challenge infection in a mouse-B. schroederi model. There was a significant reduction (62.91%) of mice vaccinated with rBs-Ag3 coupled with Freund's complete adjuvant (FCA) in recovery of challenged B. schroederi L3 compared with either controls or mice vaccinated with FCA alone. Our data indicate recombinant Bs-Ag3 may be a potential target as a vaccine antigen for giant panda ascariasis.

Lagochilascaris minor: experimental infection of C57BL/6 and BALB/c isogenic mice reveals the presence of adult worms.

The nematode Lagochilascaris minor is the causative agent of lagochilascariosis, a human disease that affects the neck region causing exudative abscesses with eggs, larvae, and adult parasites. Mice are currently considered intermediate hosts for the parasite. To determine the pattern of infection and the possibility of mice as definitive hosts for L. minor, experimental lagochilascariosis was studied in two distinct isogenic mouse strains: BALB/c and C57BL/6. Our results indicate that BALB/c mice are more resistant to L. minor infection than C57BL/6, having less intense lesions in the lungs, a lower number of nodules with encysted larvae and fewer adult worms, and displaying a higher serum level of IFN gamma. Both mouse strains had low levels of serum IL-10. We also observed adult parasites in both mouse strains, raising the possibility that mice are definitive hosts of L. minor. This is the first description of adult parasite development of L. minor in mice.

Full recovery from Baylisascaris procyonis eosinophilic meningitis.

Infection by Baylisascaris procyonis is an uncommon but devastating cause of eosinophilic meningitis. We report the first case-patient, to our knowledge, who recovered from B. procyonis eosinophilic meningitis without any recognizable neurologic deficits. The spectrum of illness for this organism may be wider than previously recognized.

Human antibody recognition of Anisakidae and Trichinella spp. in Greenland.

High levels of total IgE are observed among children in Greenland. To evaluate the extent to which Anisakidae and Trichinella spp. contribute to the high total IgE level, an ELISA and a western blot were developed for the detection of IgG antibodies to Anisakidae, based on excretory/secretory antigens from Anisakidae larvae. Western blots with Anisakidae and Trichinella antigens discriminated between Anisakidae and Trichinella infections, enabling cross-reactivity between the two parasite infections to be eliminated. Serum samples from 1012 children in Greenland were analysed for specific antibodies to Anisakidae and Trichinella. Eleven children were IgG-positive for Trichinella and nine were IgG-positive for Anisakidae, indicating a relatively low prevalence of both infections among children in Greenland. Faecal samples from 320 children were also examined for other intestinal parasites. Enterobius vermicularis was found in one sample and Blastocystis hominis in 32 samples, but no other intestinal parasites were identified. In total, 304 children had elevated total IgE levels. There was a significant association between Trichinella seropositivity and high levels of total IgE, but not between Anisakidae seropositivity and total IgE. The data indicate that parasitic infections alone do not explain the high level of total IgE observed among children in Greenland.

Larva migrans by Baylisascaris transfuga: fatal neurological diseases in Mongolian jirds, but not in mice.

Raccoon roundworms (Baylisascaris procyonis) and other Baylisascaris species cause patent or latent larva migrans (LM) in a variety of mammals and birds, including humans. It is not clear whether LM by Baylisascaris transfuga, roundworms of bears, is associated with clinical neurological disorders. To clarify this issue, ICR and BALB/c mice as well as Mongolian jirds (Meriones unguiculatus) were orally inoculated with 2,000-5,000 embryonated eggs of B. transfuga. In mice, the ascarid caused symptomatic LM of limited extent and duration, whereas the infection was fatal in jirds; i.e., they exhibited general signs such as severe depression and emaciation on days 8-11 postinfection (PI) and died, or they developed progressive and fatal neurological disorders after day 14 PI. Histological examination showed B. transfuga larvae in the brain of all mice and jirds examined, and the larvae collected from them developed to a size comparable with that of B. procyonis. There existed, however, critical differences in host reactions against larvae localized in the brain of mice and jirds; B. transfuga larvae found in mice were surrounded by granulomatous reactions and immobilized, whereas larvae found in jirds were free from any host reaction and mobile, causing extensive malacia.