We have developed a novel enzyme-linked immunosorbent assay (ELISA) based on excretory/secretory antigens of second instar Gasterophilus for the diagnosis of gasterophilosis in grazing horses. Between January 2007 and January 2009, two experiments were carried out on free-ranging horses in northwest Spain. During the first year, monthly blood samples were collected from a herd of 25 horses. In the second year, a monthly serological survey was conducted for a total of 398 different horses. All the sera were analyzed by ELISA using excretory/secretory antigens from Gasterophilus intestinalis (GphiL2ES) and Gasterophilus nasalis second-stage larvae (GphnL2ES). Climatic data were collected between January 2007 and January 2009 from local meteorological automated stations to establish the weather pattern in the study area. Observations of Gasterophilus eggs on the horses' hair and third instars passed in the faeces were also done. The kinetics of IgG response decreased against GphiL2ES from January to July, increased slowly from August and rose up to January. After a slight decrease in January, the absorbances against GphnL2ES reduced from April to August, when the lowest values were observed. The IgG values rose until the end of the study in January. Third instars were observed in the faeces in March to May, and Gasterophilus eggs were seen on the horses' hair from June to September. The highest IgG seroprevalences were achieved in winter (January-February; 100%) against both antigens. The lowest percentages of seropositivity were observed in June (3%) to the GphiL2ES, and in July (9%) to the GphnL2ES. The use of antigens from G. intestinalis second-stage larvae was shown to be suitable for diagnosing infestation by G. intestinalis or G. nasalis. We concluded that under oceanic climate conditions, the egg-laying period occurs from late spring, and eggs and first instars are found in the mouth in early summer. During summer the second instars move into the stomach and intestine, where the third-stage larvae remain until the end of winter, when pupation takes place. The adult horse bot fly emerges in the spring. Two treatments for the control of gasterophilosis are suggested: a curative in the summer to eliminate the first instars and a preventive in the autumn to suppress the second instars.
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