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clinical signs, such as ataxia and staggering (Cawdell-Smith et al. 2010), and recovery is possible only after removal from the infected paddock. The clinical manifestations of ergot poisoning in mares are early embryonic mortality, abortions, dystocia, and thickened or retained placental membranes (Fayrer-Hosken et al. 2008). Dystocia and retained placenta in three mares on the farm in question pointed towards ergot intoxication. In the present episode, clinical cases began to appear after 1 year of feeding with pearl millet fodder in large amounts. Horses new to the farm with exposure of less than 1 year did not show clinical signs and their serum GGT levels remained in the normal reference range. Observations made during the present episode are in agreement with those of Adams et al. (1993) that mycotoxins accumulate over a long time and that animals exposed to low levels of mycotoxins do not die or show clinical signs in the early phase. Likewise, in experimental studies, ponies exposed to aflatoxins for a short period of time did not develop pathognomonic signs (Asquith et al. 1980; Aller et al. 1981). In domestic animals, mycotoxin contamination reduces growth (CAST 2003), which matches the growth retardation observed in the young horses in the present episode. The liver is the primary target site for mycotoxin and


aflatoxin intoxication (Greene and Oehme 1976; Angsubhakorn et al. 1981; Vesonder et al. 1991). Upon necropsy examination, the consistency of the liver was firm, with marked centrilobular hepatic necrosis, along with the massive proliferation of


fibroblasts observed on


histopathological examination in all cases. The level of aflatoxins required to induce clinical signs in horses varies. Greene and Oehme (1976) observed that aflatoxin levels of 58.4 ppb induced severe hepatic cirrhosis, weight loss, jaundice and mortality, while Angsubhakorn et al. (1981) have reported that peanut meal containing 200 ppb aflatoxins caused fatty liver, haemorrhagic enteritis and mortality. Vesonder et al. (1991) reported that corn containing 130 ppb of aflatoxins induced hepatic necrosis in three horses. Hasso (2003) reported that 12.5 ppb of aflatoxin B1 in barley bran was sufficient to induce diarrhoea in Arabian horses. In the present episode, the aflatoxin level in sewan hay and concentrate feed mixture was 24 ppb. Both were continued even after discontinuation of pearl millet. This showed that aflatoxin levels in fodder up to 24 ppb without ergot were acceptable to the horses. However, aflatoxin levels 24 ppb or above were found to be deleterious to the horses when it had the simultaneous infestation with ergot. Thus, the signs observed in the present episode might be due to a cumulative effect of ergot and aflatoxins. However, the mycotoxin levels in the fodder were measured at a late stage of the episode, so the actual concentrations of aflatoxins ingested by the horses earlier might differ from the measured values. In the present episode, blood clotting was affected badly in all of the horses on the farm, so much so that it was difficult to obtain serum from the collected blood samples. Red blood cells automatically settled down without coagulation and plasma was separable without adding any anticoagulant. In human subjects, Lichen planus is a mucosal reaction to chronic liver cirrhosis (Del Olmo et al. 2000); similarly, in most of the horses in the present episode, nonhealing ulcerative lesions were present in the oral cavity, the inner side of lips, and over the mucocutaneous junctions of the lips. A mild yellowish discolouration of the eyes, mucous membranes and vaginal mucous membranes were present in eight cases, although plasma levels of total bilirubin were not


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excessively high. The peak level of serum bilirubin was 89.62 lmol/L in one case, with the major part of the bilirubin existing in unconjugated form. In horses, levels of unconjugated bilirubin can rise to six times the normal level during fasting (Gronwall and Mia 1972). The measurement of serum concentrations of GGT is a sensitive screening test for subclinical liver disease in horses (Curran et al. 1996). In the present case study, GGT was the most useful biochemical parameter to note effect of aflatoxins on horses. Levels of serum GGT in the horses that died of liver cirrhosis were up to 575 (U/L). Other horses at the farm that showed only weight loss and reduced appetite as clinical signs also had high serum GGT levels. On discontinuation of pearl millet from the fodder, serum GGT levels of most of the horses returned to normal reference levels, indicating that hepatic microsomal enzymes were no longer being induced. In the present episode, haemochromatosis was observed in liver cells. Hepatic cirrhosis along with haemochromatosis has been reported in horses (Pearson et al. 1994). Serum iron levels in the present episode were within normal limits for horses (Borges et al. 2007; Yoruk et al.2007). The saturation state of iron binding capacity ranged from 48 to 64%, and there was no source of high iron in the horse fodder. Thus, the haemochromatosis in the liver in the present episode might have occurred secondary to cirrhosis (Pearson et al. 1994). Therefore, the clinical, biochemical, necropsy and histopathological findings led us to conclude that the disease outbreak likely occurred due to the cumulative effect of ergot and aflatoxins.


Conclusions


In areas where pearl millet is used as fodder for horses, pearl millet should not be fed to the horses after the flowering stage because pearl millet at this stage is at risk of ergot and other mycotoxin infestation. Liver cirrhosis and related clinical signs of mycotoxicosis


develop after feeding the mycotoxin-infested crop (Aflatoxin levels 24–70 ppb) for at least 1–2 years. Old animals suffer first and most seriously. After the appearance of clinical signs in horses with liver


cirrhosis, the horses cannot be cured until or unless the primary cause is removed. GGT is the most important biomarker for subclinical or clinical injury of the liver due to mycotoxins.


Authors’ declaration of interests No conflicts of interest have been declared.


Ethical animal research Not applicable as it is a recording of a natural episode of clinical cases.


Source of funding ICAR-NRCE from the institutional fund.


Authorship


R. Dedar wrote the manuscript, did the biochemical analysis and recorded the clinical signs. N. Virmani carried out the histology. P. Bala collected and prepared the fodder samples to


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