Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses

Cyathostomins are important equine gastrointestinal parasites. Mass emergence of mucosal stage larvae causes a potentially fatal colitis. Mucosal stages are undetectable non-invasively. An assay that would estimate mucosal larval stage infection would greatly assist in treatment, control and prognosis. Previously, we identified two putative diagnostic antigens (20 and 25 kDa) in somatic larval preparations. Here, we describe their purification and antigen-specific IgG(T) responses to them. Western blots confirmed the purity of the antigens and showed that epitopes in the 20 kDa complex were specific to larval cyathostomins. No cross-reactive antigens appeared to be present in Parascaris equorum or Strongyloides westeri species. Low levels of cross-reactivity were observed in Strongylus edentatus and Strongylus vulgaris species. Use of purified antigens greatly reduced background binding in equine sera. These results indicate that both antigen complexes may be of use in a diagnostic assay.     

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.     

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.     

On the Use of 99Mo/99mTc Generators in the Analysis of Low Levels of 99Tc in Environmental Samples by Radiochemical Methods

The analysis of low levels of the long-lived fission product ⁹⁹Tc in environmental samples presents special challenges, particularly with respect to the selection of an appropriate and practicable chemical yield tracer. Of all the tracers available, ^99mTc eluated from ⁹⁹Mo/^99mTc generators appears to be the most practicable in terms of availability, ease of use and cost. These factors have led to an increase in the use of such generators for the provision of ^99mTc as yield tracer for ⁹⁹Tc. For the analysis of low levels (<1bq>³ or kg) of ⁹⁹Tc in environmental samples, consideration must be given to the radiochemical purity of the tracer solution with respect to contamination with both ⁹⁹Tc and other radionuclides. This paper details investigations carried out in relation to the use of ⁹⁹Mo/^99mTc generators for the provision of ^99mTc for use as yield tracer in the analysis of low levels of ⁹⁹Tc. The results of the study indicate that special care must be taken with the radiochemical purity of the ^99mTc solution. Analysis of eluates from a number of such generators indicates the presence of ¹⁰³Ru and ⁹⁹Tc in levels that could produce inaccuracies in the analysis of low levels of ⁹⁹Tc. Results are presented and the findings discussed with reference to low level ⁹⁹Tc analysis by radiochemical procedures and beta counting.     

On the Use of 99Mo/99mTc Generators in the Analysis of Low Levels of 99Tc in Environmental Samples by Radiochemical Methods

The results of spatial and temporal distribution of acidic and basic air pollutants in ambient air around Hsinchu Science-Based Industrial Park (SBIP), Taiwan during August 2000 to October 2001 are presented. The sampling was performed on 13 different sites around the SBIP for 12 hrs each every month, and a total of 195 samples were collected. The effect of geographical and meteorologicalconditions, and production volume of the SBIP on the variation of pollutant's concentration was investigated. The spatial distribution shows that comparatively higher concentration of pollutants was found on the sites, which are either low in altitude such as the WS (west-southern) part of SBIP, in the downwind location or close to the factories. In case the wind velocity was low, i.e. below 2 m s^-1, the concentration of pollutant tended to increase and became uniformly distributed around the science park. The temporal distribution shows a decrease in ambient concentration of pollutants from February to June 2001, mainly due to the decline in the production volume of the SBIP in this period. During the whole period of investigation, the concentration of most of the species (except HF) was found to be lower than the factory-surrounding air quality standard of Taiwan, but in some cases it was higher than the AALG.