Insect Immune Evasion by Dauer and Nondauer Entomopathogenic Nematodes.

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.

Characterisation of serum IgG(T) responses to potential diagnostic antigens for equine cyathostominosis.

Cyathostomins are ubiquitous parasitic nematodes of horses. These worms spend substantial periods as intestinal wall stage encysted larvae, which can comprise up to 90% of the total burden. Several million larvae have been reported in individuals. Emergence of these larvae from the gut wall can lead to life-threatening colitis. Faecal egg count tests, increasingly used by horse owners to inform anthelmintic treatments, do not correlate with the intra-host burden of cyathostomins; this represents a key gap in the diagnostic toolbox. Previously, a cyathostomin Gut Associated Larval Antigen was identified as a promising marker for the intra-host stages of infection. Here, cyathostomin Gut Associated Larval Antigen and an additional protein, Cyathostomin Immuno-diagnostic antigen, were investigated to examine their value in providing information on cyathostomin burden. ELISA analyses examined serum IgG(T) responses to recombinant proteins derived from individual cyathostomin species. Receiver Operator Characteristic curve analysis was performed on the ELISA data; proteins with the highest Area Under the Curve values were selected to test protein combinations to investigate which were the most informative in identifying the infection status of individuals. Three cocktail combinations were tested, comprising: (a) Cy-GALA proteins from two species and a Cy-CID protein from a third species (CT3), (b) Cy-GALA proteins from five species (CT5), and (c) all CT5 components, plus a Cy-CID protein from an additional species (CT6). The best predictive values for infection were obtained using CT3 and CT6, with similar values achieved for both. Proteins in CT3 are derived from the most commonly reported species, Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus longibursatus. This combination was selected for future development since it represents a more commercially viable format for a diagnostic test.

Preferential infectivity of entomopathogenic nematodes in an envenomed host.

Entomopathogenic nematodes and parasitoid wasps are used as biological control agents for management of insect pests such as the Indian meal moth, Plodia interpunctella. The parasitoid wasp Habrobracon hebetor injects a paralytic venom into P. interpunctella larvae before laying eggs. A previous study reported that the entomopathogenic nematode Heterorhabditis indica preferentially infects P. interpunctella that have been envenomed by H. hebetor while results in this study showed a similar preference by the entomopathogenic nematode, Steinernema glaseri. We therefore tested four hypotheses for why nematode infection rates are higher in envenomed hosts: (1) elevated CO2 emission from envenomed hosts attracts nematodes, (2) paralysis prevents hosts from escaping nematodes, (3) volatile chemicals emitted from envenomed hosts attract nematodes and increase infection, and (4) reduced immune defenses in envenomed hosts increase nematode survival. Results showed that envenomed P. interpunctella larvae emitted lower amounts of CO2 than non-envenomed larvae. Physical immobilization of P. interpunctella larvae did not increase infection rates by S. glaseri but did increase infection rates by H. indica. Emissions from envenomed hosts were collected and analyzed by thermal desorption gas chromatography/mass spectrometry. The most abundant compound, 3-methyl-3-buten-1-ol, was found to be an effective cue for S. glaseri attraction and infection but was not an effective stimulus for H. indica. Envenomed P. interpunctella exhibited a stronger immune response toward nematodes than non-envenomed hosts. Altogether, we conclude that different mechanisms underlie preferential infection in the two nematode species: host immobilization for H. indica and chemical cues for S. glaseri.

The Imaginal Disc Growth Factors 2 and 3 participate in the Drosophila response to nematode infection.

The Drosophila imaginal disc growth factors (IDGFs) induce the proliferation of imaginal disc cells and terminate cell proliferation at the end of larval development. However, the participation of Idgf-encoding genes in other physiological processes of Drosophila including the immune response to infection is not fully understood. Here, we show the contribution of Idgf2 and Idgf3 in the Drosophila response to infection with Steinernema carpocapsae nematodes carrying or lacking their mutualistic Xenorhabdus nematophila bacteria (symbiotic or axenic nematodes, respectively). We find that Idgf2 and Idgf3 are upregulated in Drosophila larvae infected with symbiotic or axenic Steinernema and inactivation of Idgf2 confers a survival advantage to Drosophila larvae against axenic nematodes. Inactivation of Idgf2 induces the Imd and Jak/Stat pathways, whereas inactivation of Idgf3 induces the Imd, Toll and Jak/Stat pathways. We also show that inactivation of the Imd pathway receptor PGRP-LE upregulates Idgf2 against Steinernema nematode infection. Finally, we demonstrate that inactivation of Idgf3 induces the recruitment of larval haemocytes in response to Steinernema. Our results indicate that Idgf2 and Idgf3 might be involved in different yet crucial immune functions in the Drosophila antinematode immune response. Similar findings will promote the development of new targets for species-specific pest control strategies.

Development of a recombinant protein-based ELISA for diagnosis of larval cyathostomin infection.

Cyathostomins are ubiquitous nematodes of horses. Once ingested, they can spend a substantial time as encysted larvae in the intestinal wall. The larvae can comprise up to 90% of the total burden, with up to several million worms reported in individuals. These stages can emerge in large numbers to cause life-threatening colitis. Direct methods for detection of encysted larval burdens in live horses do not exist. Previously, two antigen complexes were identified as promising markers for infection. A component of these, cyathostomin gut associated larval antigen-1 (Cy-GALA-1), was identified following immunoscreening of a complementary DNA library. Serum immunoglobulin G(T) (IgG(T)) responses to Cy-GALA-1 were shown to inform on larval infection. Sequence analysis of polymerase chain reaction products amplified from individual worms indicated that Cy-GALA-1 was derived from Cyathostomum pateratum. As cyathostomin infections always comprise multiple species, a diagnostic test must account for this. Here, segments of the Cy-gala gene were isolated from four common species, Cyathostomum catinatum, Cylicocyclus ashworthi, Cylicostephanus goldi and Cylicostephanus longibursatus, and the associated proteins expressed in recombinant form. The specificity and immunogenicity of each protein was confirmed. Each protein was assessed by enzyme linked immuno sorbent assay (ELISA) for its ability for informing on the presence of encysted larval infection and the level of burden.

Physiologic and systemic acute phase inflammatory responses in young horses repeatedly infected with cyathostomins and Strongylus vulgaris.

Migrating Strongylus vulgaris and encysted cyathostomin larvae cause a localized inflammatory response in horses. It is unknown whether these larvae elicit a systemic acute phase response (APR), evidenced by changes in serum amyloid A (SAA), haptoglobin (Hp), iron (Fe), albumin, or albumin/globulin (A/G) ratio. In this study, 28 horses were randomly allocated to receive either pyrantel tartrate or a pelleted placebo formulation in their daily feed. Concurrent with treatment, all the horses were administered 5000 pyrantel-susceptible cyathostomin infective larvae once daily, 5 days a week, for 24 weeks. Beginning in the fifth week, the horses also received 25 S. vulgaris larvae once weekly for the remainder of the study. At regular biweekly intervals, fecal samples were collected for quantitative egg counts, and whole blood and serum samples were collected for measurement of packed cell volume, total protein, albumin, globulin, A/G ratio, SAA, Hp, and Fe. On days 161-164, all the horses were euthanatized and necropsied. Samples were collected for enumeration of total luminal worm burdens, encysted cyathostomin larval populations, and migrating S. vulgaris larvae. Concentrations of Hp, Fe, and A/G ratio were associated significantly with strongyle burdens. Only treated male horses had significant increases in serum albumin. Larval S. vulgaris did not associate with Fe, whereas Fe was associated negatively with both total cyathostomin burdens and encysted L4s. The A/G ratios differed significantly between the two treatment groups. Significant differences between groups and individual time points were also observed for Hp and Fe, whereas SAA concentrations remained low throughout the study. In general, this study illustrated that experimental inoculations with S. vulgaris and cyathostomins may be associated with changes in Hp, Fe, and serum proteins, but not with SAA. Overall, these changes suggest that mixed strongyle infections elicit a mild acute phase reaction.

Characterization of the inflammatory response to anthelmintic treatment of ponies with cyathostominosis.

Cyathostomins can cause a severe inflammation of equine large intestine characterized by substantial ventral edema and pronounced protein loss. Anthelmintic treatment of horses can result in a localized inflammatory response in the colonic mucosa of clinically normal horses. The aim of this study was to evaluate the systemic inflammatory response of ponies naturally infected with cyathostomins to single dose representatives of three anthelmintic drug classes, namely, oxibendazole, pyrantel pamoate, and moxidectin. Thirty ponies aged between 1 and 18 years of age were allocated to one of three anthelmintic treatments groups. Anthelmintic efficacy was evaluated using the fecal egg count reduction test performed weekly between 2 and 8 weeks post-treatment. Inflammatory responses were evaluated on days 0, 1, 3, 5, and 14 after treatment using hematology, measurement of the acute phase inflammatory markers serum amyloid A, fibrinogen, haptoglobin, and iron, and real-time PCR measurement of expression of the genes for interleukins 1-ß and -10, tumor necrosis factor-α, and interferon-γ. There were subtle inflammatory responses to treatment, but cytokine expression was significantly associated with the interaction term between treatment group and anthelmintic efficacy (P<0.05). Of the acute phase markers, only fibrinogen associated with treatment group. The findings suggest that systemic inflammatory responses subsequent to anthelmintic treatment of cyathostomin infection are minimal. It is possible that this response is 'buffered' by anti-inflammatory products of the parasites and/or the anti-inflammatory effects of the macrocyclic lactones.

Recent advances in diagnosing pathogenic equine gastrointestinal helminths: the challenge of prepatent detection.

Parasites infecting horses are ubiquitous and clinically important across the world. The major parasitic threats to equine health are cyathostomins, Parascaris equorum, Anoplocephala perfoliata, and Strongylus vulgaris. Increasing levels of anthelmintic resistance reported world wide in equine parasites have led to recommendations of constructing sustainable parasite control programmes based on systematic surveillance of parasite levels. Regulations at the European Union level now make anthelmintics available on prescription-only basis and disallow prophylactic treatment. This emphasizes the needs for reliable and practical diagnostic tools for detection of major parasites infecting equines. The current, widely used coprological techniques are important and useful, but they do have considerable limitations as they are incapable of diagnosing the pathogenic migrating stages. Species-specific molecular assays have been developed for diagnosing patent infections with 21 cyathostomin species, A. perfoliata, and S. vulgaris, but none of these have found use in practice. An antibody-directed enzyme-linked immunosorbent assay (ELISA) has been developed, validated and made commercially available for diagnosing A. perfoliata infection, but interpretation is complicated by the fact that horses not harbouring tapeworms can maintain elevated antibody titres. Recent work with a coproantigen ELISA has shown promise for reliable detection of current A. perfoliata infection. Perhaps most remarkable is the fact that the pathogenic larval stages of cyathostomins and large strongyles cannot be detected by any of the available diagnostics. With the lengthy prepatency periods characterizing these parasites, there is a huge need for developing such assays. The recent identification of a possible diagnostic marker for encysted cyathostomins holds great promise, and could become very useful in clinical practice. Several attempts have been made to construct assays for diagnosing the highly pathogenic migrating larvae of S. vulgaris, but none of these have performed sufficiently to make a useful test. The present review illustrates that classical coprological techniques remain the cornerstone of equine parasitology diagnosis and surveillance, and will remain so in a foreseeable future. However, promising progress has been made for developing assays capable of diagnosing prepatent stages of strongyle infection, and there is reason to hope for validated and useful assays in the relative near future.

Isolation of potentially useful antigens from cyathostomin third-stage larvae by using a fast protein liquid chromatography one-step method.

Three major protein complexes (51, 29, and 15 kDa, named P1 to P3, respectively) were resolved by gel filtration of the excretory/secretory antigens collected from a mixture of horse cyathostomin third-stage larvae (L3s). The potential application for the detection of infected horses was assessed with an enzyme-linked immunosorbent assay (ELISA) by the comparison of the serological and copromicroscopical results. The value of the area under the receiver operating characteristic (ROC) curve was higher than 0.9 when the three peaks were used. Elevated values (>90%) for the sensitivity, specificity, and the positive-likelihood ratio were also observed for all the antigen complexes. A significant increment in the IgG antibody levels 4 weeks prior to the observation of eggs in the feces of weanlings naturally infected was recorded. Our results indicate that the evaluation of chemotherapy is possible by using immunoenzymatic probes and fast protein liquid chromatography (FPLC)-purified antigens. Data collected in the present investigation indicate that FPLC isolation offers a very helpful one-step method for collecting antigens with diagnostic potential to be employed in immunoenzymatic probes.

Characterization of local immune response against lungworms in naturally infected sheep.

This study describes the immunohistochemical and histochemical phenotypes of inflammatory cells in sheep lungs infected with lungworms. A total of 20 naturally infected sheep lungs were used. Protostrongylus spp., Muellerius capillaris, Neostrongylus linearis, and Cystocaulus ocreatus were the chief organisms determined from such lesions, which were of a chronic nature. All the lungs had many developmental stages of the parasites and a similar inflammatory response, which included numerous mast cells, eosinophils, T cells, B cells, dendritic cells, and macrophages. In the bronchial and interstitial tissues, the inflammatory cells were dominated by MHCII, CD1, CD4, CD5, CD14, CD21, IgM, and CD172a positive cells, whereas CD2 and WC1 positive cells were detected less. The data provided additional evidence that subsets of inflammatory cells were included within ovine lungs infected with lungworms; however, understanding the entire immune-response process and development of resistance to lungworms in sheep remain to be clearly elucidated.

Evidence of Parelaphostrongylus tenuis infections in free-ranging elk (Cervus elaphus) in southern Ontario.

The antemortem detection of a Parelaphostrongylus tenuis infection in a free-ranging wild elk (Cervus elaphus) in southern Ontario is documented. Postmortems on other free-ranging elk that died during 2000-2005 indicated that 59% (17/29) were infected with P. tenuis, based on presence of lesions in the brain.

Cytokine responses to Cyathostominae larvae in the equine large intestinal wall.

To investigate cytokine responses in cyathostomin infection, we quantified mucosal interleukin-4 (IL-4), interleukin-10 (IL-10), tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma by reverse transcriptase-competitive polymerase chain reaction. The analysis was performed on large intestinal wall samples obtained from six anatomical sites spanning the caecum and colon of 17 naturally exposed horses. The numbers of developing larvae (DL) and early third stage larvae (EL3) were ascertained using transmural illumination and pepsin digestion techniques, respectively. Levels of each cytokine transcript were correlated with local intestinal wall burdens of Cyathostominae larvae. IL-4 and IL-10 levels showed significant correlations with EL3 and DL burdens at several sites. No significant correlations were observed with IFNgamma. A pro-inflammatory response, typified by detection of TNFalpha transcript, was observed at a few sites in some horses with inflammatory enteropathy associated with emerging or emerged larvae. However, this cytokine was measured at an insufficient number of sites to enable statistical analysis. Levels of IL-4, IL-10 and IFNgamma transcript were compared between two groups: one group consisting of horses with low to high mucosal burdens (Group A) and the other, of horses with negative/negligible mucosal burdens (Group B). Significant differences in IL-4 (P<0.001) and IL-10 (P<0.001) transcript levels were observed between the groups, with higher levels observed in Group A. No significant differences in IFNgamma were observed. Taken together, these results indicate that Th2 responses predominate in mucosal Cyathostominae infection prior to larval reactivation.

A dot-blot ELISA comparable to immunoblot for the specific diagnosis of human parastrongyliasis.

A dot-blot enzyme-linked immunosorbent assay (dot-blot ELISA) using an electroeluted 31-kDa glycoprotein from adult worms of Parastrongylus cantonensis as the specific antigen was evaluated for the immunological diagnosis of patients infected with P. cantonensis. The sensitivity and specificity for the detection of serum antibody to P. cantonensis in dot-blot ELISA were both 100%, as determined with serum samples of ten P. cantonensis-infected patients, 60 patients with other related parasitic infections, and 20 uninfected controls. The test was as sensitive and specific as the immunoblot test which revealed a reactive band of 31 kDa. Both the dot-blot ELISA and immunoblot detected all sera from ten P. cantonensis-infected individuals, but not with those of other heterologous parasitoses (gnathostomiasis, toxocariasis, filariasis, paragonimiasis, cysticercosis and malaria) or sera from healthy controls. The dot-blot ELISA is much simpler to perform than the immunoblot technique, and the test can be applied under field conditions where sophisticated facilities are lacking.

Recent developments in research into the Cyathostominae and Anoplocephala perfoliata.

Intestinal helminths are an important cause of equine disease. Of these parasites, the Cyathostominae are the commonest group that infect horses. These nematodes consist of a complex tribe of 51 species, although individual horses tend to harbour 10 or so common species, in addition to a few rarer species. The Cyathostominae can be extremely pathogenic, and high levels of infection result in clinical symptoms ranging from chronic weight loss to colic, diarrhoea and death. As part of their life cycle, immature cyathostomins penetrate the large intestinal wall, where they can enter a state of inhibited larval development. These larvae can exist in this state for months to years, after which they subsequently re-emerge. If larvae re-emerge in large numbers (i.e. several million), severe pathological consequences ensue. The inhibited larvae are also relatively refractory to several of the currently available anthelmintics, so that horses treated previously with anthelmintics can still carry life-threatening burdens of these parasitic stages. Little is known about the cyathostomin larvae during their mucosal phase, and current research efforts are focused on investigating the biology of these stages. Much of the research described here highlights this area of research and details studies aimed at investigating the host immune responses that the mucosal larvae invoke. As part of this research effort, molecular tools have been developed to facilitate the identification of larval and egg stages of cyathostomins. These molecular tools are now proving very useful in the investigation of the relative contributions that individual, common cyathostomin species make to the pathology and epidemiology of mixed helminth infections. At the more applied level, research is also in progress to develop an immunodiagnostic test that will allow numbers of mucosal larvae to be estimated. This test utilises antigen-specific IgG(T) serum antibody responses as markers of infection. As anthelmintic resistance will be the major constraint on the future control of the Cyathostominae, researchers are now actively investigating this area and studies aimed at elucidating the molecular mechanisms of drug resistance are described. Another parasite which has assumed a clinically important role in horses is the tapeworm, Anoplocephala perfoliata. This parasite is prevalent world-wide and has been shown to be a significant cause of equine colic. Because previous methods of estimating the infection intensity of tapeworm were inaccurate, recent research has been directed at developing an immunodiagnostic ELISA for these cestodes. Specific IgG(T) responses to antigens secreted by adult tapeworms have been shown to provide a reasonable indication of infection intensity. An ELISA based on these responses is now commercially available. The steps involved in the development of this ELISA are described here. In addition to these recent advances in research, this review also outlines the principle areas for future research into these important equine parasites.

Equine cyathostomins.

This collection of articles provides an in depth account of five presentations delivered during the Symposium on Equine Cyathostomins held at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP), New Orleans, Louisiana,10­14 August 2003. The symposium was organized and chaired by Ray M. Kaplan and Jacqui B. Matthews and focused on new developments in two major areas of current importance: the immunobiology of cyathostomin­horse interactions and anthelmintic resistance.

Identification of antigens with potential for immunodiagnosis of Parelaphostrongylus tenuis and Elaphostrongylus cervi infections in red deer (Cervus elaphus elaphus).

Red deer (Cervus elaphus elaphus) were infected experimentally with Parelaphostrongylus tenuis in New Brunswick, Canada, and with Elaphostrongylus cervi in New Zealand. Excretory-secretory (E-S) antigens from adult P. tenuis were evaluated for their serodiagnostic potential in identifying P. tenuis and heterologous E. cervi infections in a Western blot. The antigen recognition profile of sera from animals infected with P. tenuis varied between individuals and with duration of infections, whereas that of pooled sera from animals infected with E. cervi showed less variation. A single molecule of 42-43 kDa was recognized consistently by sera from all animals infected with either P. tenuis or E. cervi. Sera from unexposed control deer and from those with other heterologous nematode infections did not consistently identify this antigen. Serorecognition of the 42-43-kDa antigen by deer infected with P. tenuis resulted in a sensitivity of 99% and a specificity of 85% (> or =1 mo postinfection). Although antibody to this antigen waned with time, the persistence of recognition up to 34 mo postinfection with P. tenuis exemplifies its diagnostic value. The sensitivity and specificity of diagnosis using this molecule were each 100% for identifying deer infected with E. cervi (> or =3 mo postinfection). Two other molecules from E-S of adult P. tenuis, 26-28 and 10-12 kDa, were also diagnostic, although their recognition was not persistent throughout infections. These 2 molecules may prove useful in combination with the 42-43-kDa antigen to help identify all infected animals during all phases of infections. This research represents the first conclusive identification of antigens with real potential for reliable antemortem immunodiagnosis of both P. tenuis infections and heterologous E. cervi infections.

Mast cell and eosinophil mucosal responses in the large intestine of horses naturally infected with cyathostomes.

From December 1998 to March 2000, caecum and ascendant colon of 42 horses naturally infected with cyathostomes were collected during routine necropsy or from a local slaughterhouse. Changes in the numbers of mucosal and submucosal mast cells (MMC and SMMC), intraepithelial, mucosal and submucosal eosinophils (IE, ME and SME) in the large intestine were investigated by histochemical techniques in relation to the worm burdens. The effect of age was examined in three subgroups: 6-24-month-old horses (group 1), 2-10-year-old horses (group 2) and horses more than 10 years of age (group 3). No globule leucocytes were detected in any sections. No significant variations with breed or sex were observed in cell counts. The main variations were higher eosinophil counts in groups 2 and 3 and a marked increase of the MMC counts in the oldest horses (group 3). For each cell type, the infiltration was homogeneous and generalised along the large intestine. In the whole horse sample, the IE numbers were the only parameters that correlated with the MMC and SMMC counts. Very few significant relationships were found between mast cells and eosinophils in groups 1 and 3, whereas numerous positive correlations were recorded in group 2. In the whole horse sample, several correlations were found between different cell counts and cyathostome burdens. The numbers of larvae, adult worms, and the total worm burdens were related to some of the tissular eosinophil counts while the percentage of early third stage larvae (EL3) was linked to mast cell densities. These relations between cells and worm populations showed variations with age. In group 1, most of the significant associations were found between eosinophil counts (IE and SME) and the total numbers of larvae and worms; in group 2, they were noticed between the three eosinophil types and the total cyathostome burdens. In group 3, a MMC hyperplasia was observed and correlations were mostly recorded between these MMC and the total numbers of adult worms or the percentage of EL3. Several associations were also detected between eosinophils (mainly ME and/or IE) and different cyathostome burdens. These variations in the relationship between inflammatory cells and cyathostomes seemed to be consistent with the cellular changes observed among the three age groups. These results suggest that eosinophil and mast cell infiltrations quantified in the large intestine wall might be associated with cyathostome infection.

An aspartyl protease inhibitor orthologue expressed by Parelaphostrongylus tenuis is immunogenic in an atypical host.

Parelaphostrongylus tenuis is a neurotropic nematode common in white-tailed deer (Odocoileus virginianus) of eastern North America. This parasite is the causative agent of a debilitating neurologic disease in atypical hosts, including domestic livestock. In order to identify proteins of potential significance in the host-parasite relationship, a cDNA library was produced from adult P. tenuis mRNA. Screening the library with antisera from infected red deer (Cervus elaphus elaphus) and immunized AO strain rats, we identified clones with sequence similarities to aspartyl protease inhibitors from several parasitic nematodes. Antibody that was generated against this recombinant protein of P. tenuis (Pt-API-1) detected the native protein in E/S products, in muscle and gonad, and on the surface of the cuticle of adult male and female P. tenuis. The native protein was detected in internal structures of first-stage (L1) and third-stage (L3) larvae. Reverse transcription-PCR confirmed expression of Pt-api-1 in L1, L3, and adult male and female worms. Expression of Pt-API-1 throughout the life cycle of P. tenuis suggests an essential function. Antibodies specific for recombinant Pt-API-1 were detected by enzyme-linked immunosorbent assay in sera from 12 red deer experimentally infected with P. tenuis. Antibodies were detected within 28 to 56 days postinfection. Responses were sustained or biphasic in animals with patent infections, consistent with expression of Pt-API-1 by L1. Our results are compatible with findings in other parasitic nematodes showing that aspartyl protease inhibitors are highly immunogenic.

Antigen-specific IgG(T) responses in natural and experimental cyathostominae infection in horses.

Equine clinical larval cyathostominosis is caused by simultaneous mass emergence of previously inhibited larvae from the mucosa of the colon. Clinical signs include diarrhoea, colic, weight loss and malaise, and in up to 50% of cases, the disease results in death. Cyathostominae spend a large part of their life cycle as larval stages in the intestinal mucosa. Definitive diagnosis is difficult due to the lack of diagnostic methods for pre-patent infection. In the present study, the enzyme-linked immunosorbent assay (ELISA) was used to investigate isotype responses to larval cyathostominae somatic antigen. Measurement of anti-larval IgG(T) responses appeared to have the most immunodiagnostic potential. An increase in IgG(T) response was detected to crude larval antigen by 5 weeks post-infection (PI) in individual infected ponies. Subsequently, IgG(T) responses to larval and adult somatic extracts were examined by Western blotting using sera from experimentally-infected horses and helminth-naive animals (n=6). Two antigen complexes, designated A and B, in larval somatic antigen were recognised specifically by the infected animals by 7 weeks PI. Sera taken from 23 endemically-infected animals, whose cyathostominae burdens had been enumerated, were also used to identify putative diagnostic antigens. Eighteen horses had positive mucosal worm burdens (range 723-3,595,725) and all but two of these animals had serum IgG(T) antibody specific to either complex. Moreover, IgG(T) responses specific to antigen complexes A and B were absent in all five parasite negative horses that were tested. Serum IgG(T) responses to either of the two complexes were identified in five clinical cases tested. IgG(T) responses to adult antigen somatic extracts were more heterogeneous, with no clear pattern between experimentally-infected ponies and helminth-free controls. The results indicate that increases in serum IgG(T) to mucosal larvae occur in the pre-patent period and that two antigenic complexes within somatic preparations of these stages have immunodiagnostic potential.

Considerations for the control of equine cyathostomes in arid areas.

Internal parasites of horses are ubiquitous but that does not suppose that the level of infection does not vary with climatic conditions. Climate determines the limits of where a parasite species can survive the external environment and weather determines the transmission pattern within the climatic bounds [Levine, N.D., 1963. Adv. Vet. Sci. 8, 215-261]. Arid areas have a more limited exposure potential to important parasites but the level of exposure can nonetheless lead to disease. It must be remembered that, even in arid areas, it does rain and irrigation, overflow from water troughs, dew dripping off buildings and on the vegetation can also provide the medium to allow escape of larval cyathostomes from feces to forage. How horses earn their living is most important in determining the level of exposure to cyathostomes. Recreational grazing, which surely does more for the soul of the owner than for the nutrition of the horse, almost absolutely insures that horses will encounter larvae. To be certain, in arid areas there may be an opportunity for horses to spatially separate grazing and dunging areas but not all horses are so disposed, and even if they are they may not be able to do so.