Abstract: Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n = 46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n= 19) or H. muscae (n =4), and others (n = 7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3.3 %) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100 % and a sensitivity of approximately 97 % (being able to specifically detect as little as approximately 0.02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae.

Abstract: REASONS FOR PERFORMING STUDY: Sandy soil is often mentioned as a risk factor in the development of sand-related gastrointestinal disease (SGID) in the horse. There are other variables, but few studies confirm any of these. OBJECTIVE: To investigate soil type, pasture quality, feeding practice in the paddock, age, sex and body condition score as risk factors for sand intake in the horse. METHODS: Faeces were collected from 211 Icelandic horses on 19 different studs in Denmark together with soil samples and other potential risk factors. Sand content in faeces determined by a sand sedimentation test was interpreted as evidence of sand intake. Soil types were identified by soil analysis and significance of the data was tested using logistic analysis. RESULTS: Of horses included in the study, 56.4% showed sand in the faeces and 5.7% had more than 5 mm sand as quantified by the rectal sleeve sedimentation test. Soil type had no significant effect when tested as main effect, but there was interaction between soil type and pasture quality. Significant interactions were also found between paddock feeding practice and pasture quality. CONCLUSION: To evaluate the risk of sand intake it is important to consider 3 variables: soil type, pasture quality and feeding practice. Pasture quality was identified as a risk factor of both short and long grass in combination with sandy soil, while clay soil had the lowest risk in these combinations. Feeding practice in the paddock revealed feeding directly on the ground to be a risk factor when there was short (1-5 cm) or no grass. Also, no feeding outdoors increased the risk on pastures with short grass, while this had no effect in paddocks with no grass. More than 50% of all horses investigated in this study had sand in the faeces. POTENTIAL RELEVANCE: The identification of risk factors is an important step towards prevention of SGID. Further research is necessary to determine why some horses exhibit more than 5 mm sand in the sedimentation test and whether this is correlated with geophagic behaviour.

Abstract: Most studies published to date that used fecal glucocorticoid measurements to assess adrenocortical activity in primate (and many nonprimate) species applied a specific cortisol or corticosterone assay. However, since these native glucocorticoids are virtually absent in the feces of most vertebrates, including primates, the validity of this approach has recently been questioned. Therefore, the overall aim of the present study was to assess the validity of four enzyme-immunoassays (EIAs) using antibodies raised against cortisol, corticosterone, and reduced cortisol metabolites (two group-specific antibodies) for assessing adrenocortical activity using fecal glucocorticoid metabolite (GCM) measurements in selected primate species (marmoset, long-tailed macaque, Barbary macaque, chimpanzee, and gorilla). Using physiological stimulation of the hypothalamo-pituitary-adrenocortical (HPA) axis by administering exogenous ACTH or anesthesia, we demonstrated that at least two assays detected the predicted increase in fecal GCM levels in response to treatment in each species. However, the magnitude of response varied between assays and species, and no one assay was applicable to all species. While the corticosterone assay generally was of only limited suitability for assessing glucocorticoid output, the specific cortisol assay was valuable for those species that (according to high-performance liquid chromatography (HPLC) analysis data) excreted clearly detectable amounts of authentic cortisol into the feces. In contrast, in species in which cortisol was virtually absent in the feces, group-specific assays provided a much stronger signal, and these assays also performed well in the other primate species tested (except the marmoset). Collectively, the data suggest that the reliability of a given fecal glucocorticoid assay in reflecting activity of the HPA axis in primates clearly depends on the species in question. Although to date there is no single assay system that can be used successfully across species, our data suggest that group-specific assays have a high potential for cross-species application. Nevertheless, regardless of which GC antibody is chosen, our study clearly reinforces the necessity of appropriately validating the respective assay system before it is used.

Abstract: We investigated the impact of enclosure size and animal density on behavior and adrenocortical secretion in Pere David's deer in Dafeng Nature Reserve, China. From February 15 to April 16 in 2004, we conducted two experiments. First, we studied maintenance behavior and conflict behavior of Pere David's deer stags in a large enclosure (200 ha) with low animal density (0.66 deer/ha) and a small display pen (0.75 ha) with high animal density (25.33 deer/ha). The maintenance behavior we recorded included standing, locomotion, foraging and rest. During the behavioral observations, we collected fresh voided fecal samples from the stags periodically, and analyzed the fecal cortisol concentrations in those samples using radioimmunoassay technique. Second, we monitored the fecal cortisol concentrations of one group of stags (12 deer lived in an enclosure of 100 ha) before and after transferred into a small pen (0.5 ha). We found that in the first experiment: (1) there were significant differences in standing and rest whereas no significant differences of locomotion and foraging between the free-ranging group and the display group; (2) frequency of conflict behavior in the display group was significantly higher than those in the free-ranging group; and (3) fecal cortisol concentration of the display group (326.17+/-16.98 ng/g dry feces) was significantly higher than that of the free-ranging group (268.98+/-15.21 ng/g dry feces). In the second experiment, there was no significant difference of the fecal cortisol concentrations among sampling days, but the mean fecal cortisol concentration of the day after transferring (337.46+/-17.88 ng/g dry feces) was significantly higher than that of the day before transferring (248.44+/-7.99 ng/g dry feces). Comparison with published findings, our results indicated that enclosure size and animal density affect not only behaviors, but also adrenocortical secretion in Pere David's deer. Small living space with high animal density may impose physiological stress to captive Pere David's deer. Moreover, long-term physiological stress and increase of conflict behavior may subsequently affect survival and reproduction of the deer.

Abstract: Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species.