Clinico-pathological studies of imidacloprid toxicity and its amelioration with vitamin C in broiler chickens Komal1, Gupta R.P.1, Lather Deepika1,* 1Department of Veterinary Pathology, College of Veterinary SciencesLala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, Haryana, India *Corresponding author: e-mail: deepikalather@yahoo.co.in
Abstract An experimental study was conducted on 120 broiler chicks to investigate the clinic-pathological changes of imidacloprid (IMC) toxicity and to assess the effect of vitamin C supplementation on the toxicity. Chicks at the age of 7 days were divided into two groups (A and B). The birds of group A were given vitamin C @100 mg/kg body weight orally daily, throughout the experiment. At the age of 14 days, the birds of groups A and B were further divided into two subgroups (A1, A2 and B1, B2). Birds of group Aland Blwere given IMC @ 5.206 mg/kg body weight (1/5th of maximum tolerated dose) orally till the end of experiment. The chicks of group B1 exhibited clinical signs such as depression, decreased appetite, excessive salivation, watery diarrhea, closing of eye and respiratory distress. Group B1 showed significantly lower mean body weight. Histopathological lesions in IMC treated groups were hepatitis accompanied with fatty change and necrosis of hepatocytes, bronchiolitis, myocarditis, enteritis, proventriculitis, depletion of lymphocytes in lymphoid organs, interstitial nephritis and degeneration of Purkinje cells in cerebellum. It is concluded that IMC administration caused immunosuppressive, circulatory and inflammatory lesions which were ameliorated significantly with vitamin C supplementation. Top Keywords Chicken, Imidacloprid toxicity, Vitamin C. Top |
INTRODUCTION Insecticides are applied on floor and walls of the poultry houses, also on equipments within the house or in some cases directly on the birds as vapours, dust or spray leading to the contamination of the external and internal milieu of birds. Indirect exposure of insecticides to the birds occurs through the use of insecticide contaminated poultry litter1 and feed constituents having insecticide residue in poultry ration2. Imidacloprid (IMC) a neonicotinoid insecticide is most widely used insecticide because it has high selectivity for insects and apparently safe for humans3. Imidacloprid causes almost complete and virtually irreversible blockage of post-synaptic nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS) of insects. Studies on pathological effects of imidacloprid administration in poultry are few, and there is no report on ameliorative effect of vitamin C supplementation on imidacloprid (IMC) toxicity in chickens. Therefore, the present study was undertaken to elucidate the pathology due to imidacloprid toxicity in chickens and its amelioration by vitamin C supplementation. |
Top MATERIALS AND METHODS Experimental design One hundred and twenty chicks at the age of 7 days were divided randomly into two groups (A and B) and these birds were vaccinated with Newcastle disease vaccine (Lasota strain) via intranasal route. Birds of group A were given vitamin C @ 100 mg/kg body weight orally for 28 days4. At the age of 14 days, the birds of group A and B were further divided into two subgroups (A into A1 and A2; B into B1 and B2). Birds of group A1 and B1 were given imidacloprid @1/5th of maximum tolerated dose (5.206 mg/kg body weight) orally through drops, daily throughout the experiment. Birds of group B2 were kept as control i.e. without any treatment. Birds in different groups were observed daily for clinical signs and mortality, if any and were also weighed weekly. Six birds of each subgroup were sacrificed at the start of the experiment and subsequent on 7th, 14th, 21st and 28th day post treatment and post mortem examination was conducted for gross lesions. Representative tissues of the different organs such as liver, heart, lungs, kidneys, intestine, proventriculus, pancreas, brain, spleen, thymus and bursa of Fabricius was collected in 10 per cent buffered formalin for histopathological studies. The formalin fixed tissues were processed for paraffin embedding technique. The tissues were properly trimmed, washed in running tap water, dehydrated in graded ethyl alcohol, cleared in benzene and embedded in paraffin wax (melting point 60–62°C). Sections of 3–4 μm thickness were cut using automatic microtome and stained with haematoxylin and eosin5. Statistical analysis Duncan Multiple Range Test as modified by Kramer6 at 5 per cent level of significance using SPSS 16.0 version software was used to analyse the data of body weight. Individual means were compared for statistical significance using least significance difference (P<0.05). Top RESULTS Clinical signs and mortality In groups A1 and B1, the chicks exhibited clinical signs such as depression, decreased appetite, excessive salivation (Fig. 1), dullness, watery diarrhea, closing of eye and respiratory distress after 10–15 min of administration of IMC and persisted for 1–2 hours. However, the decrease in appetite was noticed in group B1 throughout the experiment. These clinical signs of imidacloprid toxicity were of mild intensity in vitamin C supplemented group. No mortality was observed in any of the experimental groups. Body weight Mean body weights of chicks in different experimental groups at different intervals are presented in Table 1. The mean body weight of group B1 (imidacloprid treated) was lower than group B2(control) throughout the experiment but the difference was significant (P ≥ 0.05) only on day 28 post treatment. The mean body weight of group A1 (vitamin C supplemented and imidacloprid treated) was also lower than control group B2 but higher than group B1 though the difference was non-significant at different intervals indicating ameliorating effect of vitamin C on the body weight. Pathological studies No gross and histopathological changes were observed in any of organs of birds in groups A2 and B2 (control groups) at different intervals. Characteristic pathological changes in different organs of imidacloprid treated groups at different intervals are depicted in Table 2. Imidacloprid treated group (B1) Gross lesions: Chicks of group B1 exhibited congestion in various organs such as liver, kidney (Fig. 2), lung, spleen, heart, intestine and pancreas (Fig. 3) from 7 days post treatment (DPT) onwards. The severity of congestion was increased on subsequent days post treatment. However, the severity of congestion was almost similar on days 21 and 28 post treatment. From 14 DPT onwards, there were hemorrhages in spleen, liver (Fig. 4), intestine and lungs. Liver showed necrotic areas on 21 DPT onwards. Histopathological lesions Liver revealed congestion in central vein and sinusoids along with a few lymphocytes in portal triad areas from 7DPT onwards. These lesions became more severe on subsequent intervals. Besides, hyperplasia of bile duct, hemorrhages and fatty changes were also observed from 14 DPT onwards. Focal area of necrosis accompanied with infiltration of lymphocytes and heterophils was noticed from 21 DPT onwards (Fig. 5). Heart revealed hemorrhages and congestion in myocardium on 7 DPT onwards. Focal myocarditis characterized by infiltration of mononuclear cells and few heterophils was also observed on 14 DPT onwards. Lung revealed congestion and hemorrhages from 7 DPT onwards. There was bronchiolitis characterized by hyperplasia of mucosal epithelium and peribronchiolar infiltration of lymphocytes along with perivascular oedema from 14 DPT onwards. Kidney revealed congestion both in medulla and cortex including glomerular capillaries from 7 DPT onwards. There were nephrotic lesions in renal tubular epithelium along with mild hemorrhages from 14 DPT onwards. Focal interstitial nephritis characterized by mononuclear cells infiltration along with atrophy of renal tubules (Fig. 7) was noticed from 21 DPT onwards. Intestine exhibited enteritis characterized by infiltration of lymphocytes and few heterophils in lamina propria along with hemorrhages and congestion in serosa from 14 DPT onwards. Mild proventriculitis characterized by congestion and infiltration of lymphocyte in mucosa was observed from 14 DPT onwards. Pancreas showed congestion in exocrine portion from 14 DPT onwards. Spleen, thymus and bursa of Fabricius revealed congestion and hemorrhages along with depletion of lymphocytes (Fig. 9). There was congestion in molecular and granular layers and degeneration or necrosis of few Purkinje cells (Fig. 10) characterised by loss of dendrites and nucleus in cerebellum from 21 DPT onwards. Vitamin C supplemented imidacloprid treated group (A1) Gross lesions: Congestion and hemorrhages in various organs was also noticed in group A1 but it was of mild degree as compared to group B1 at different intervals post treatment. Liver showed necrotic foci at few places on 21 and 28 DPT. Histopathological lesions Hepatic lesions exhibited in group A1 were of lower magnitude as compared to group B1. Liver revealed mild congestion (Fig. 6) on 7 DPT onwards. From 14 DPT onwards, hyperplasia of bile duct, dilatation of sinusoids, hemorrhages and fatty changes were also observed but these lesions were less severe as compared to group B1. No cardiac lesions were observed on 7 DPT. However, there was mild hemorrhages and congestion along with infiltration of mononuclear cells and few heterophils at few places in myocardium from 14 DPT onwards. Congestion and hemorrhages in lungs were less severe as compared to B1 group at different intervals. Peribronchiolar infiltration of lymphocytes was noticed from 14 DPT onwards. However, perivascular oedema was not seen in group A1. Kidney revealed mild congestion (Fig. 8) from 7 DPT onwards. Other lesions such as nephrosis and interstitial nephritis were not noticed. Enteritis and proventriculitis were noticed. Pancreas showed congestion from 14 DPT onwards but it was less severe as compared to B1 group. Spleen, thymus and bursa of Fabricius revealed mild congestion and hemorrhages. Cerebellum revealed congestion in molecular and granular layers along with degeneration or necrosis of few Purkinje cells characterized by loss of dendrites and nucleus from 21 DPT onwards. Top DISCUSSION The clinical signs noticed due to IMC administration within very short period might be due to its rapid and complete absorption7,8. Similar clinical signs have also been observed by other workers9,10,11-12 in experimental toxicity of imidacloprid in chicken, rat and mice. Vitamin C has been reported to increase feed intake and lower the plasma corticosterone13,14 which might have resulted in improvement of body weight. |
The pathological changes are in accordance with the findings of other workers in chickens11, quails15 and rats16,17-18 who have also reported degeneration, necrosis and circulatory lesions due to administration of IMC. The liver plays central role in the biotransformation and disposition of xenobiotics19. The liver has been reported to be the principal target organ of IMC toxicity10,11. Nevertheless, the kidney plays important role in the detoxification for many xenobiotics and is frequently susceptible to the nephrotoxic effects11. Nephrotoxic lesions due to IMC toxicity has also been reported by other workers in chickens11 and rats16. No report could be traced in the literature in animals and poultry regarding the effect of IMC toxicity on gastrointestinal tract pathology. |
It is interesting to note that there were neurotoxic lesions particularly degeneration and necrosis of few Purkinje cells in cerebellum due to IMC toxicity in the present study. Such lesions16,20 have also been reported by other workers in rats. The accumulation of IMC and its metabolites in brain has been reported in mice21. Histopathological results of lymphoid organs in chicken revealed depletion of lymphocytes indicating immunosuppressive effect of IMC toxicity. Almost similar findings have been reported22 in chickens administered with IMC @5 mg/kg body weight for 45 days. |
Supplementation of vitamin C revealed that the lesions in the different organs due to IMC toxicity were of mild magnitude. More or less similar results have been reported in quails15 and in rats18, particularly in IMC- induced hepatic lesions. Vitamin C plays an important role in hepatotoxicity due to insecticides by preventing the effect of free radicals on hepatocytes23. No report could be traced on effect of vitamin C on IMC toxicity in chickens. However, partial ameliorating effect of vitamin C supplementation on pathological changes in different organs due to chlorpyrifos toxicity has been reported in chickens4. |
On the basis of overall results observed in the present study it is concluded that supplementation of vitamin C @ 100 mg/kg body weight caused reduction in toxicity of imidacloprid as evidenced by its ameliorating effects on clinical signs and pathological changes. |
Top Figures | Fig. 1.: (Group B1, 21 DPT): Bird showing excessive salivation
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| | Fig. 2.: (Group B1, 14 DPT): Severe congestion in kidney (arrow)
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| | Fig. 3.: (Group B1 and A1, 14 DPT): B1- Congestion of intestine and pancreas, A1- Mild congestion in intestine and pancreas
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| | Fig. 4.: (Group B1, 14DPT): Congestion and haemorrhage (arrow) on liver.
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| | Fig. 5.: (Group B1, 28DPT): Liver revealed necrosis associated with lymphocytic infiltration. H&E ×400
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| | Fig. 6.: (Group A1, 28DPT): Liver revealed mild congestion in sinusoids. H&E ×200.
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| | Fig. 7.: (Group B1, 28DPT): Kidney revealed focal interstitial nephritis characterized by infiltration of lymphocytes causing atrophy of renal tubules (arrow). H&E ×200
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| | Fig. 8.: (Group A1, 28DPT): Kidney revealed mild congestion. H&E ×400
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| | Fig. 9.: (Group B1, 28DPT): Bursa of Fabricius revealed karyorrhexis and pyknotic nuclei indicating necrosis of lymphocytes (arrow). H&E ×400
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| | Fig. 10.: (Group B1, 21DPT): Cerebellum revealed necrosis of Purkinje cells (arrow). H&E ×400.
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Tables | Table 1: Mean body weight (g) in different experimental groups at different intervals (Mean±SE).
| | Groups | Post treatment interval (Days) | | 0 | 7 | 14 | 21 | 28 | | A1 | 391.67a±5.57 | 703.83a±15.41 | 1189.20a±36.86 | 1742.50a±72.08 | 2123.20ab±29.46 | | A2 | 408.83a±4.47 | 813.83b±32.9 | 1365.80b±53.43 | 2062.50b±49.12 | 2290.00b±91.25 | | B1 | 404.17a±5.20 | 702.33a±21.83 | 1168.30a±56.31 | 1718.30a±66.21 | 2015.00a±59.14 | | B2 | 390.33a±8.98 | 791.33ab±40.46 | 1240.00ab±48.95 | 1910.80ab±67.53 | 2270.00b±97.02 |
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| a,b: Means with unlike superscript in the column differ significantly (P < 0.05) | | | Table 2.: Comparison of characteristic pathological changes (gross and histopathology) in different organs of imidacloprid treated groups at different intervals.
| | Pathological changes | Group B1 | Group A1 | | 7 DPT | 14 DPT | 21 DPT | 28 DPT | 7 DPT | 14 DPT | 21 DPT | 28 DPT | | 1. Congestion in various organs | + | ++ | +++ | +++ | + | + | ++ | ++ | | 2. Hemorrhage in various organs | - | ++ | +++ | +++ | - | + | ++ | ++ | | 3. Hepatic degeneration/necrosis | - | + | +++ | +++ | - | + | + | ++ | | 4. Myocarditis | - | + | ++ | +++ | - | + | ++ | ++ | | 5. Bronchiolitis | - | + | ++ | +++ | - | + | ++ | ++ | | 6. Focal interstitial nephritis | - | + | ++ | +++ | - | - | - | + | | 7. Enteritis | - | + | ++ | +++ | - | + | + | ++ | | 8. Proventriculitis | - | + | ++ | ++ | - | + | + | ++ | | 9. Lymphocytic depletion in lymphoid organs (spleen & bursa) | - | + | ++ | +++ | - | + | + | ++ | | 10. Purkinje cell degeneration/necrosis | - | - | + | ++ | - | - | + | + |
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| + mild, ++ moderate, +++ severe | |
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