Clinical symptoms
Feeding experiments demonstrated that the small-tailed Han sheep appeared to have a toxic reaction after ingestion of E+ A. inebrians, including absent-mindedness, blank stares and stumbling, but not following ingestion of E- A. inebrians or the control feed. At 35-d one small-tailed Han sheep exhibited poisoning symptoms including mydriasis, neck stiffness, limb tic, nasal mucosa bleeding, and weak breathing. It suffered a hypotensive crisis and its condition worsened, with muscle flaccidity, loss of defecation, abdominal swelling, retention of urine, and ultimately death. These symptoms are similar to the toxicity symptoms of ergonovine and ergine [9,10,11]. Ergonovine poisoning can cause excitement, muscle relaxation and allergic reaction, apparent intelligence depression, gastrointestinal dysfunction and stomach ache. Ergine can cause anesthesia and retroperitoneal fibrosis, urinary tract obstruction and renal failure. Therefore, it was concluded that the sheep fed E+ A. inebrians were poisoned by the alkaloids that are produced by the N. gansuense endophyte symbiont of A. inebrians.
The feed intake of sheep fed E+ A. inebrians was significantly less than those fed E- A. inebrians or the control group, so the endophytic fungi reduce either palatability or feeding drive of the animals, or possibly both. In the first three days of the experiment, the feed intake reduction of the E+ group was very large but then rose slightly, suggesting initial aversion of the sheep to feed containing endophye infected drunken horse grass, followed by gradual acclimation. This is consistent with prevously published findings [12].
Suppression of feed intake of small tailed han sheep offered endophyte infected A. inebrians is consistent with observed body weight loss, so the endophyte infected A. inebrians can be considered a factor in poor body condition of small tailed han sheep. This is consistent with a research report of Blaney [13].
Endophyte infected A. inebrians fed to sheep resulted in significantly elevated levels of urine ketone bodies and occult blood and increased numbers of animals with acidic urine. Ketone bodies are a product of the decomposition of fatty acids, and include acetoacetate, acetone and ß- hydroxybutyric acid [14,15,16]. When their production is greater than the liver can metabolize, the ketones will accumulate to produce acidosis. Under physiological conditions, ketone bodies cannot be detected in animal urine, but they can appear with long-term malnutrition, hunger, long-term anesthesia and after a wound. Ergot alkaloids produced by E+ A. inebrians have effects on the nervous system, with the increased presence of ketone bodies possibly being responsible for long-term anesthesia, but the specific mechanism of action needs further research.
Occult blood is hemoglobin or red blood cells in urine which cannot be observed by the naked eye directly [17, 18]. The bleeding of different parts of the urinary tract, due to hemolytic disease, poisoning [19] or blood transfusion reactions [20], can cause a positive occult blood reaction. In this study, the poisoning of sheep by feeding E+ A. inebrians was very strongly correlated with the positive occult blood reaction, and can be considered a probable cause.
The pH of animal urine is affected by feed properties, and generally herbivore urine is slightly alkaline, but some nutritional metabolic diseases such as ketonemia, or pathogenic heat or malnutrition can make urine acidic [16, 21]. In this study, the observed acidic urine may have been caused by ketone bodies, but the mechanism is still ill-defined.
Liver and renal function
Serum ALT and AST values are used mainly for the detection of liver disease in livestock, and generally the ALT and AST activity of sheep ranges from 25.0 to 70.0 U/L, 40.0 to 123.0 U/L respectively [22]. In the present study, the activities of serum ALT and AST in E+ sheep were significantly higher than the E- or Control sheep. These results for the E+ group were similar to those prevously published [23,24,25,26].
Creatinine is a substance associated with energy metabolism of muscles, and elevation occurs when its discharge is increased. Serum creatinine concentration rises rapidly early in kidney disease [27]. That is consistent with the detected significant increase in the first week of the experiment. The normal range of Serum creatinine is usually less than 150 mmol/L. Reduced creatinine levels are of no clinical significance, but when levels of 250 mmol/L or more are observed, this may inidcate kidney dehydration or heart failure [28].
Relative weight of organ
E+ A. inebrians ingestion brought about higher of brain, liver, lung and kidney, but lower that of the uterus. This result would be expected, given the negative effects in liver and renal function, and more loss of body weight outlined above.