Ochratoxin A (OTA), mainly produced by Aspergillus ochraceus has been reported to be more toxic than even aflatoxin B₁¹⁴. Besides adverse effects on kidneys, the target organ for OTA toxicity, it has been incriminated to induce genotoxicity, teratogenisity, carcinogenisity, immunotoxicity and neurotoxicity. OTA being a feed contaminant causes variable toxicity in different species. It has been implicated in fatal human kidney disease, Balkan endemic nephropathy. In India, OTA has been reported to be a common contaminant in various commodities⁴,⁶,⁷,¹¹. Most of the spontaneous cases of ochratoxicosis in the field have either been underreported or remained undiagnosed. Inspite of voluminous work carried out on different aspects of ochratoxicosis, sequential clinico-pathological studies employing graded doses of OTA are limited. The present investigation was undertaken to assess the sequential changes in certain clinico-pathological parameters in Wistar rats fed a diet containing graded levels of OTA, upto six weeks.

Freeze dried culture of Aspergillus ochraceus NRRL- 3174, procured from National Centre for Agricultural Utilisation and Research (NCAUR), Peoria, Illinois, USA, was employed for OTA production as per the method described by Trenk et al.²⁰. OTA was estimated using thin layer chromatography and UV-Vis Spectrophotometer (Genesys™ 10, Thermo Electron Corporation, Pittsford, USA) at 333 nm against the standard toxin procured from Sigma Chemicals Limited, USA and got confirmed from Animal Feed Analytical and Quality Control Laboratory (AFAQCL), Veterinary College and Research Institute, Namakkal, Tamil Nadu.

Weaned Wistar rats of either sex, approximately 21 days of age were procured from Laboratory Animal Resource Section, IVRI, Izatnagar and were maintained on a formulated feed for rats (tested to be free of aflatoxin B₁ and ochratoxin A) obtained from Feed Technology Unit of IVRI. Rats housed in polypropylene cages were provided with rice husk as the bedding material. All the animals had free access to standard laboratory animal diet and clean water. All the procedures, conducted on the experimental animals were duly approved by the Institutional Animal Ethics Committee (IAEC) and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

After an acclimatization period of 7 days, all the animals were weighed again and randomly assigned to four groups of 30 rats each so as to give approximately equal initial group mean body weights. The experimental animals were given the following group-wise treatments. Gr. I received basal diet tested free of OTA and acted as the control. Gr. II, III and IV received diets containing 1, 2 and 4 mg OTA/kg of feed, respectively. The experimental diets were provided daily ad lib for six weeks. Ten animals from each group were sacrificed at 2, 4 and 6 weeks post intoxication.

Animals were closely observed twice daily for development of clinical signs. Body weights of the animals were recorded at weekly intervals. The rats were euthanised humanely by deep inhalation anaesthesia in a CO₂ chamber. The heart blood samples were collected quickly after the sacrifice in dry sterilized vials containing anticoagulant EDTA (ethylene diamine tetra acetic acid, 1mg/ml). Blood glucose was estimated using glucometer (Ascensia Entrust, Bayer Health Care LLC, USA) with blood glucose test strips (Bayer) as per manufacturer's instructions in randomly selected 6 rats from each group. The haematological parameters such as haemoglobin concentration (Hb), packed cell volume (PCV), total erythrocyte count (TEC), total leucocyte count (TLC) and differential leucocyte count (DLC) were carried out as per standard procedures ⁵. The values of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were derived from the values of Hb, PCV and TEC⁵. Blood (2–3 ml) was collected separately in a dry, clean and sterilized test tube and allowed to clot. The sera were harvested and stored at - 20°C. The sera samples were analysed for the total proteins (Biuret method) and albumin (BCG dye binding method) using standard kits of (Glaxo, Qualigens Diagnostics) and the values of globulins were derived by subtracting values of albumin from total proteins.

All data were presented as Mean + SEM. Two -way analysis of variance (ANOVA) was used to detect differences among groups and within groups and the means were compared by Dunnett's Multiple Comparison test. All analyses were performed with GraphPad InStat software (San Diago, USA); and a value of P ≤ 0.05 was taken as significant ¹⁷.

Even though extensive studies have been carried out on ochratoxicosis, the reports on sequential clinicopathological alterations employing graded doses of OTA are meager in rats. The clinical signs of dullness, weakness, reduced feed intake, progressive decrease in growth rate, intermittent diarrhoea and increased water intake observed in OTA treated groups were also described earlier⁸, which were neither specific nor of diagnostic significance. The increased water intake was reflective of renal damage¹. Increased water intake has been reported in diabetogenic animals¹⁸. Similar mechanism might be playing some role in the pathogenesis of ochratoxicosis as the serum glucose levels were significantly higher especially in rats receiving OTA at 4 ppm levels in the present study. Increased water intake was in consonance with the earlier findings⁷, ¹³, ²¹ which might be to compensate water loss in diarrhoeic animals recorded in the present study.

There was no mortality in control animals, whereas 4 rats (13.3 %) died in 4 ppm group, two during 4^th week and two during 6^th week of experimentation. At 2 ppm OTA level, 2 rats (6.6%) died, one in the 5^th week and one in the 6^th week and at 1 ppm level, 3 animals (10 %) died in the 3^rd week. The mortality ranging from 6.6 to 13.3 % in a dose related fashion in OTA treated rats have also been recorded earlier¹⁵. The mortality in rats of OTA fed groups could be due to fatal kidney damage and hepatotoxicity as observed histopathologically¹⁰.

Significant reduction in body weights (Table 1) in treated groups occurred from 3^rd week onwards, the lowest recorded at 6 weeks in 4 ppm OTA group (105.40±2.15 g vs. 143.00±5.19 g in control). Growth depression observed in treated groups could be attributed to reduced feed intake⁷. However, hypoproteinaemia as observed in the present study and reported by other workers¹⁵ as well as increased protein loss through urine leading to hypoproteinaemia¹³ might have led subsequently to progressive decrease in body weights. It has been earlier reported that severity of toxic effect of OTA in animals increased when fed with protein-deprived ration¹⁶

The haemoglobin, TEC and PCV levels were found decreased in the toxin treated groups suggesting anaemia (Table 2), which became pronounced as the experiment progressed. This might be due to degenerative changes in bone marrow, spleen and liver as was observed earlier in rats¹⁵ and rabbits⁷. The anaemia, microcytic hypochromic type observed in the present study might be due to impaired haemoglobin synthesis owing to Fe²⁺ chelating property of OTA¹². Decreased feed intake and hypoproteinemia might also be correlated with the development of anaemia.

In the present investigation, significant leucocytopenia due to lymphocytopenia was observed. The findings were supported by earlier workers in different species of animals in ochratoxicosis⁷,¹⁵,¹⁹. The increase in relative neutrophil counts in a dose dependent manner was due to lymphocytopenia which were also recorded in rats³ due to ochratoxicosis.

Hyperglycemia noticed, particularly at 4 ppm level at 6 weeks post feeding was in agreement with those reported in rats¹⁸ in OTA toxicity. The hyperglycemia might be due to the diabetogenic effect of OTA¹⁸. Contrary to it, OTA was found to specifically inhibit phosphoenol pyruvate carboxykinase (PEPCK) enzyme in pigs, which blocked the gluconeogenesis and was responsible for reduced serum glucose⁹.

Biochemically, OTA was found to cause hypoproteinemia as evidenced by decrease in serum total proteins, albumin and globulin levels (Table 3) in a dose and time dependent manner which was possibly due to hepatotoxicity ¹⁵, ²¹. OTA inhibited the protein synthesis in the liver, major site of protein synthesis, possibly through competitive inhibition of phenylalanine t-RNA synthetase². Anorexia, reduced feed intake and inadequate absorption due to OTA induced damage in gastrointestinal tract might also have contributed to hypoproteinemia. The prominent OTA induced damage to nephrons resulting in proteinuria especially albuminuria¹³ might also have contributed to development of hypoproteinemia. Based on the present study it was concluded that OTA caused significant sequential alterations in clinical and haematobiochemical parameters in Wistar rats in a dose and time dependent manner.

Means bearing at least one common small letter superscript (a, b, c, d) in a column and capital letter superscript (A to E) in a row do not differ significantly (P<0.05) n=10. Group I: control; Group II:1ppm OTA; Group III: 2ppm OTA; Group IV: 4ppm OTA

Means bearing at least one common small letter superscript (a, b, c, d) in a column and capital letter superscript (A to D) in a row do not differ significantly (P<0.05) n=6; Group I: control; Group II:1ppm OTA; Group III: 2ppm OTA; Group IV: 4ppm OTA; N.B. No significant differences were noticed in the values of eosinophils, basophils and monocytes in different groups at various intervals

Means bearing at least one common small letter superscript (a, b, c, d) in a column and capital letter superscript (A to D) in a row do not differ significantly (P<0.05) n=6; Group I: control; Group II: 1ppm OTA; Group III: 2ppm OTA; Group IV: 4ppm OTA

REFERENCES