MOLECULAR AND ULTRASTRUCTURAL PATHOLOGY OF THIRAM (TMTD - TETRA METHYL THIURAM DISULFIDE) INDUCED TIBIAL DYSCHONDROPLASIA (TD) IN BROILERS
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Date
2011-08
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Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P
Abstract
ABSTRACT :
Tibial dyschondroplasia (TD) is a major metabolic cartilage disease of young poultry, in which the tibial growth plate cartilage (TGPC) fail to undergo osteogenic transition leading to the retention of thickened white opaque a vascular cartilage plug at the end of the proximal tibia and tibio-tarsal bones. Thiram is a systemic fungicide used as a seed protectant and is available in different forms dust, flowable and wettable powder. Thiram is the model fungicide which induces the TD in broilers.
Four hundred and twenty (420) day-old male broilers chicks were wing banded, individually weighed and distributed into twelve groups of 35 chicks each, reared under identical managemental conditions in separate battery brooder pens for a period of 35 days. Group I was fed with basal diet served as absolute control, group II and III were fed with 60 ppm and 100 ppm of TMTD. Group IV, V and VI were fed with 200 ppm CuSo4, LivOrdain-FS and USCura Tox-FS at the rate of 1g/kg diet as respective positive controls. Group VII and VIII were fed with 60 ppm and 100 ppm of TMTD and 200 ppm of CuSo4 as an ameliorative agent. Where as group IX and X were fed with 60 ppm and 100 ppm of TMTD and LivOrdain-FS at the rate of 1g/kg diet, XI and XII groups were fed with USCura Tox-FS at the rate of 1g/kg diet and daily observed for clinical signs. Weekly weight gains and feed conversion ratio (FCR) were statistically analyzed and found significantly (P≤ 0.01) lower weight gains and higher FCR values in TMTD treated groups.
Six birds from I, III, IV, VII and X were sacrificed on 5th, 10th and 15th day and TGPC paired samples were collected for gene (s) expression (QRT-PCR), ultrastructural pathology (TEM & SEM) and immunohistochemistry. Further, six birds from each group sacrificed at end of 1st, 3rd and 5th week, and blood was collected for haemato-biochemical studies. Tibial bones, liver and kidney samples were also collected for histopathological and ultrastructural studies.
The results were statistically analyzed. Significantly (P≤0.01) lower values were found in TEC, Hb, PCV, TLC and DLC during 1st, 3rd and 5th week of experiment in respective groups (II, III, IV, VIII, XI, X and XII). The results revealed that TMTD exhibited a partial suppressive action on erythrpoiesis, leucopoiesis and Hb synthesis in addition to causation of severe TD lesions.
Serum biochemistry showed significantly (P≤ 0.01) lower values of glucose (IV, VII and IX), cholesterol (II, III and X), total protein and A/G ratio (III, IV, IX and XI) during the experimental period. The lower values of AST and ALT were recorded in groups II, IV, VI, VII, IX and X. The GGT and ALP values were lower in groups V, IX, XI, XII and IV, V and XII respectively. Lower calcium levels were observed in groups II, VIII and XII. Overall the ameliorative agents did not show significant effect on haemato-biochemical parameters.
The regulation of different TD specific genes was studied in this experiment. Significantly (P≤ 0.01) lower CT values of VEGF, VEGFR1 and Bcl-2 were recorded in groups III, VIII and X. Lower CT values of MMP2 was observed in group V, VIII and X, where as MMP3 it was observed in groups IV, VIII and X during 5th ,10th and 15th day of experiment. Up and down regulation of respective genes are playing a vital role in causation and repair of TD lesion.
Tibial bone, liver and kidney weights, length and diameter of tibial bones were calculated. They revealed highly significant lower values in TMTD treated birds when compared with other groups.
The graded lesions (+, ++ and +++) of longitudinally sectioned tibial growth plate cartilage showed higher score at its widest point in TMTD treated samples over other groups.
The changes like shrinkage, congestion of liver and kidneys were moderate to sever in group II and III, mild in group VII, VIII, X and XII and mild enlargement of the organs were observed in group IV, V and VI than group I.
The TGPC revealed marked changes in hypertrophic zone than the other zones. Based on severity the lesions were classified as mild (+), moderate (++) and severe (+++). Mild lesions were characterized by slight thickening of proliferating zone, transitional zone, and hypertrophic zone and were characterized by pyknotic nucleoli, disintegrating nucleolus and empty clusters of chondrocytes. The moderate lesions were summarized as pyknotic, chromatolytic, degenerating nucleus and the chondrocytes are ovoid without nucleus with more of eosinophilic matrix. Severe lesions were characterized by grater thickening of proliferating zone, grater thinning of hyaline zone, absence of chromatin material and empty clefts like chondrocytes are grouped in clusters.
These graded lesions were prominent in group II, III, XII and followed by group VII, X, VIII, IX and XI during 5th,10th and 15th day and 1st, 3rd and 5th week of experimental period.
The sections also revealed pyknotic nuclei, empty (in few), oval and large disorganized clusters of chondrocytes in group III. Proliferating blood vessels, reorganization of chondrocytes with dark dens centrally placed nucleus with few empty cells were observed in group IV, where as group V showed dilated vessels and clusters of chondrocytes are in rows as that of normal. Group VIII sections showed large oval empty chondrocytes, with more of matrix, few with pyknotic nuclei. The sections of group X were large oval chondrocytes with nucleus, more of matrix and cells are in the form of clusters than rows.
Liver and kidney samples from all the groups were studied. Severe dilation of CV, mild dilatation of sinusoids, mild to moderate fatty change, hydropic degeneration of perivascular area and shrunken hepatocytes, with thickened wall of the blood vessels and karyorrhexic and pyknotic nuclei observed in group III samples. Dilation of sinusoids, moderate congestion of CV, moderate fatty change and focal aggregates of MNC’s were observed in groups III, VIII and X samples. Mild to moderate lesions were revealed in group IV and V.
The kidney sections of group III showed moderate to severe lesions like intertubular dilatation, shrunken glomeruli. Group IV sections showed grater dilatation of tubules, shrunken glomeruli, hyaline casts and mild degeneration of tubular epithelium. Mild to moderate intertubular haemorrhages, focal areas of cystic dilatation, shrunken glomeruli, degenerating tubular epithelium were observed in group V. Group VIII sections revealed moderate cystic dilatation of tubules, degenerating tubular epithelium. Focal aggregates of mononuclear cells, focal areas of hyaline casts, greater intertubular dilatation was observed in group X samples. The changes were mild in early age and pronounced as the treatment advanced.
Immunohistochemistry of TGPC was studied in groups I, III, IV, V, VIII and X for detection of specific anti apoptotic (Bcl-2) antigen which was detected in the chondrocytes of groups III, VIII and X with varied stages of intensive reaction of antigen. Group I, IV and V were revealed without reaction.
Expression of VEGF was done for detection of endothelial cellular changes of blood vessels by using monoclonal antibodies against VEGF antigen. Increased intensity of reaction was found in group III and IV and mild staining reaction was also observed in groups V, VIII and X.
The TGPC, liver and kidney samples from different groups (I, III, IV, V, VIII and X) during 5th, 10th and 15th day were used for transmission and scanning electron microscopy (TEM and SEM).
The TEM lesions in hypertrophic zone of TGPC were characterized by dilatation and vesiculation of rough endoplasmic reticulum (RER), enlargement of para-nuclear space, and swollen mitochondria with electron-dense flocculent material, loss of matrix and dilatation of Golgi saccules and nuclear chromatin margination which is indicative of apoptosis was observed in group III and VIII. In few sections o group III increased the lucency of cytoplasm of necrotic chondrocytes were also found. Group IV and V the changes were consisted of a well developed ribbon shaped RER with dilated regions in the vicinity of Golgi complex and secondary vacuoles. Group X revealed round nucleus, with faint nucleolus mild dilated RER, dense mitochondria was observed. Chondrocytes of 15th day revealed large lipid inclusions, vesiculated and disarranged stacks of rough ER along with apoptotic cells which had cytoplasmic crescentric cap like structures of condensed chromatin is indicative of apoptosis and early cell death.
The TEM of liver sections of group II and III revealed moderate to severe changes like margination of chromatin, disrupted nucleolus, and swollen mitochondria with loss of matrix, cytoplasmic vaccuolation was observed. Mild to moderate changes were also observed in group IV and V. Group VIII samples revealed shrunken hepatocytes with pyknotic nucleus and scanty chromatin margination, complete loss of cellular architecture, cytoplasmic vaccuolation distorted nuclear membrane and loss of other organelle.
The kidney samples of group II and III revealed moderate to severe lesions like swelling of nucleus, margination of chromatin, condensed and vacuolated mitochondria, and narrow tubular lumen. Group IV and V showed mild to moderate changes with the presence of brush boarder appearance in PCT. Samples of group VIII revealed shrunken tubules with electron dense numerous fat bodies, pyknotic nucleus, margination of chromatin, cytoplasmic vacuoles and vacuolated mitochondria. Group X samples showed distorted and necrotic epithelial cells with pyknotic nuclei, condensed nucleolus, loose inter cellular junction, swollen mitochondria, loss of brush boarder and narrowed tubular lumen.
The SEM lesions of tibial bone growth plate cartilage of group III revealed necrotic areas of cartialginous structures and complete loss of haversion sysytem group IV sample showed a moderate alteration of haversion system and also the haemorrhges were observed. Group V cartialge sample showed a narrowed haversion system but appeared to be normal. The VIII and X group samples revealed completely altered haversion system with moderate haemorrhges were observed.
The liver samples of group III revealed moderate to severe dilatation of CV with perivesicular space and necrotic parenchyma. Thin slices of group IV samples showed severe dilatation of blood vessels altered hepatic parenchyma, in group V revealed an altered parenchyma, congestion and haemorrhages.The samples of group VIII exhibited severe dilataion of blood vessels and necrosis of hepatic parenchyma. Group X slices were revealed a mild dilataion of vessels and altered hepatic architexure.
The kidney samples of group III revealed moderate swelling of tubules with completely closed lumen and haemorrhages, the group IV showed an intertubular dilatation, a mild swelling tubules and narrowed tubular lumen. Severe haemorrhages and narrowed tubules were observed in group V. Group VIII samples were showed a moderately swollen tubules with complete closure of tubular lumen, a mild intertubular dilatation was seen among group X samples.
On perusal of literature limited information is available about TMTD induced TD pathogenesis at molecular, ultrastructural level and ameliorative agents. The present proved that the TMTD is a potential fungicide to induce TD at 60 ppm and 100 ppm and an attempt was made with different ameliorative agents to counter act the TMTD effect. The ameliorative agents which were used in this experiment did not repair TD lesion completely, up and down regulation of TD specific genes, apoptotic and other sub-cellular changes like RER dilation, changes in the mitochondrial structure and nucleus, specific antigen reaction in immunohistochemistry and graded histopathological lesions of TD affected chondrocytes were moderate to severe in groups II and III, mild to moderate in groups IV, VIII and X and mild in other groups comparatively control.
Preliminary attempt has been made to study the cellular and sub cellular changes in liver and kidney due to its vital role in detoxification and excretion of metabolites and toxic substances. The changes were pronounced in TMTD treated groups followed by CuSo4 and other ameliorative groups. Thiram caused damage to the liver and kidney. The body weight gains and FCR was excellent in LivOrdain, CuSo4 and US CuraTox groups over TMTD treated groups.
The result of this study high lighted the importance of further investigation in this area to minimize the economic losses due to TD in broilers.