Pathology of combined effect of ochratoxin A and citrinin in rabbits

The present investigation was designed to study the sub-chronic effects of OTA and CTN both alone and in combination. New Zealand White rabbits, divided into 4 groups, were fed basal rations containing OTA@ 0.75 ppm (Group I), CTN @ 15 ppm (Group II) and OTA @ 0.75 ppm + CTN@ 15 ppm (Group III) and standard toxin free feed (Group IV) for 60 days. The rabbits were assessed for clinical signs, haematology, serum biochemistry, tissue damage by oxidation, immune responses, apoptosis in kidneys and spleen, hormone levels, gross and histopathological changes. Clinically, toxicosis was evident in the toxin fed groups from second to third week onwards with symptoms characterised by anorexia, decreased body weights, lethargy and dehydration. Mortality was observed in all the toxin fed groups, with highest mortality (12.5%) in groups I and III. Body weights were significantly reduced from fifth week onwards with significant decrease in the combination group among the toxin fed groups. Significant haematological changes characterised by microcytic and hypochromic anaemia and leucocytopaenia due to lymphocytopaenia and relative neutrophilia were found in all the toxin fed groups. Biochemically, hypoproteinemia, hypoal- buminemia and hypoglobinemia and increased values of creatinine, AST, ALT, ALP and LDH were found in all the toxin fed groups. A maximum effect was observed in the combination group. SOD, catalase and MDA activities were increased in groups I and III suggesting damage due to oxidative stress and free radicals. Significant decrease in the antibody titres to SRBCs in all treated groups and decrease in CMI responses as evaluated by DTH reaction and LTT assay were observed in groups I and III. Increase in the number of apoptotic cells in flowcytometry and definite ladder patterns of DNAs in the agarose gel electrophoresis were observed in the toxin treated groups. Grossly, kidneys were the most affected organs in all the groups followed by liver, small intestine, spleen and brain, and testes in group I. Histopathologically, kidneys showed PCTs degeneration, irregularly dilated tubules with presence of casts in the lumen, increased periglomerular spaces, dilatation of DCTs, vacuolations and congestion in glomeruli in all the groups. In liver, hepatocytic degeneration, bile duct epithelial hyperplasia and hypertrophy, peribiliary fibrosis alongwith frequent karyomegaly and binucleation were found in the treated groups. Brain revealed congestion and haemorrhages in meninges in the toxin fed groups and increased perivascular spaces in the gray matter of cerebral cortex. Intestines revealed increased goblet cell activity in all the groups and depletion of lymphocytes in Peyer's patches in groups I and III. These changes were most severe in the combination treatment. In testes, severe degeneration of spermatogonial cells with complete cessation of spermatogenesis was observed in the OTA fed rabbits. Ultrastructural studies in kidney suggested PCT mitochondria to be most sensitive to mycotoxin injury and confirmed apoptotic changes in the kidney in OTA and combination groups. In the present study, additive effect of both toxins was observed based on clinical, haematological, biochemical, immunological, pathomorphological and ultrastructural changes. OTA and CTN even in subclinical doses also produced severe toxic effects.