Hematobiochemical and pathological alterations due to chronic chlorpyrifos intoxication in indigenous chicken.

OBJECTIVE: The present study investigates the effect of oral administration of chlorpyrifos (CPF) in indigenous chicken.
MATERIALS AND METHODS: The birds were divided into two groups I and II. Group I served as control and group II was treated with CPF (0.36 mg/kg) orally daily up to 12 weeks. Blood samples were assayed for hemoglobin (Hb), total erythrocyte count (TEC), total leukocyte count (TLC), differential leukocyte count, and biochemical constituents like alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholinesterase (CHE), total protein and uric acid. Representative pieces of tissues from liver and kidney were collected weekly for histopathological examination.
RESULTS: A significant (P < 0.01) increase of Hb, TEC, TLC, and heterophil percent and decrease of lymphocyte percent was observed. Serum ALP, AST, ALT, and uric acid increased significantly and CHE values decreased significantly in CPF treated birds. The protein level remained similar. Uric acid level was found to be increased significantly in the treated group. The results indicate that chronic CPF intoxication produces hematological, biochemical, and pathological changes in treated birds.

Introduction

Chlorpyrifos (CPF) (O, O-diethyl-O-[3, 5, 6-trichloro-2-pyridyl] phosphorothioate), an organophosphorus compound, particularly affects the cholinesterase (CHE) system. CPF affects the nervous system of the pest by inhibiting the breakdown of acetylcholine (ACh), a neurotransmitter. Resulting accumulation of ACh in the synaptic cleft causes overstimulation of the neuronal cells, which leads to neurotoxicity and eventually death.[1] Toxicological studies of CPF in chickens focused on the sub-acute effects on plasma or serum enzymes and other biochemical parameters,[2] examination of delayed neurotoxicity,[3] foodborne toxicity,[4] developmental effects,[5] and pathology of long-term exposure.[6]

In the present context, the hematological, biochemical, and pathological effect of chronic exposure to CPF was evaluated in indigenous chickens.

Materials and Methods

Animals

Three-month-old unsexed 24 indigenous chickens procured from All India Coordinated Research Project on Poultry, College of Veterinary Science (CVSc), A. A. U., Khanapara Guwahati-781022 were wing banded, weighed, and reared in the Department of Pathology, CVSc with ad libitum supply of feed and water. The experimental trials were approved by the Institutional Animal Ethics Committee (No. 770/ac/CPCSEA/FVSc/AAU/IAEC/11-12/128).

Chemical (Insecticide)

Commercial products of CPF (20%) used in this study were procured from Excel Crop Care Private Limited, Mumbai, India.

Experimental Protocol

Thirty-two chickens were randomly segregated into two groups of 16 each and fasted for 6 h prior to dosing. Group I served as control and received distilled water p.o. for 90 days. Group II served as CPF group. CPF was diluted in a tenfold serial dilution with distilled water to obtain a concentration of 0.2 mg/ml (10−4). Fresh preparations were orally administered daily using oral gavage. Group II was administered 0.36 mg/kg b.w. CPF (1.8 ml of 10−4 dilution) daily up to 90 days. Doses were calculated on weekly body weight basis and administered accordingly. The birds were closely watched for the presence of clinical signs, if any, and sacrificed at weekly interval till the end of the experiment.

Hematology

Hemoglobin (Hb), total erythrocyte count (TEC), and total leukocyte count were estimated with the help of automated hematology cell counter (Model-ms4e). Differential leukocyte count was analyzed as per standard method.[7]

Biochemical Estimations

Blood samples were collected via wing vein or jugular vein puncture at 0 day and weekly intervals up to 12th week. In control group, blood was collected at the same time and same day of collection as in case of treated group; the serum was rapidly separated and processed for determination of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), CHE, total protein and uric acid using commercial kits (Siemens Diagnostics India Ltd) on ultraviolet-visible spectrophotometer.

Histopathological Examination

Representative samples irrespective of lesions from the liver and kidney were collected weekly in 10% neutral formalin. After washing in running water and dehydration in alcohol, tissues were embedded in paraffin, and 5 μm paraffin sections cut and stained with hematoxylin and eosin as per standard method.[8]

Statistical Analysis

All data were expressed as mean ± standard error. The statistical significance of the mean differences between control and treated groups was analyzed by one-way ANOVA. Statistical calculations were performed with the SPSS 11.5 computer program (SPSS Inc., Chicago, Illinois, USA). The value of (P < 0.05) was taken as the cut-off value to consider differences statistically significant.

Results

Immediately after oral dosing the chickens developed increased thirst which disappeared gradually, except for reduced feed intake and gradual reduction in body weight gain which showed an increase of 39% and 23.1%, respectively, for the control and treated birds over a period of 12 weeks. The difference in weight gain was found to be statistically significant (P < 0.05). Birds appeared to be active and alert. After 2 months of treatment, some of the birds exhibited slightly staggering gait, leg weakness, tremor, and diarrhea. Some developed curled toes with pale mucous membrane and prominent keel bone. These symptoms disappeared subsequently towards the end of the experiment.

Effects of CPF on hematological parameters are tabulated in Table 1. There was significant (P < 0.05) increase in Hb and TEC; heterophil percent was increased, and lymphocyte percent was decreased in the treatment group compared to control. There was no variation in the monocyte, eosinophil, and basophil percent in both the groups.

Table 1

Hematological parameters (mean±SE) of control and CPF treated indigenous chicken

Alkaline phosphatase, AST, ALT, CHE, total protein, and uric acid in chickens fed CPF are presented in the Table 2. When compared to the control and CPF groups ALP, AST, and ALT values differed significantly (P < 0.05) in the CPF treated group. There was significant inhibition of CHE (P < 0.05) in CPF intoxicated chickens compared to the control chickens. Significant increase in the uric acid level was observed in the CPF treated group. The levels of total protein remained unaltered in both groups.

Table 2

Biochemical parameters (mean±SE) of control and CPF treated indigenous chicken

Histopathologically, during the 1st month of treatment, the liver of CPF treated birds showed mild congestion with scattered hemorrhages and mild mononuclear cell infiltration. By 2nd month, changes were more prominent, and consisted of degeneration of hepatocytes with focal areas of hepatocellular necrosis [Figure 1] in the CPF-treated birds. Mild to moderate proliferation of biliary epithelial cells around the portal veins with formation of new bile ducts was observed [Figure 2]. Gradually by 3rd month, changes were more severe in the CPF treated group and were characterized by dilatation of sinusoids, marked congestion, disruption of hepatic cords, focal to diffuse hemorrhage, and mononuclear cell infiltration in the hepatic parenchyma, particularly around the blood vessels of the portal region [Figure 3].

Figure 1

Liver of chlorpyrifos-treated chicken showing hemorrhage, degeneration, and necrosis during the second month of treatment (H and E, ×400)

Figure 2

Liver of chlorpyrifos-treated chicken showing proliferation of biliary epithelial cells with formation of new bile ducts during 2nd month of treatment (H and E, ×400)

Figure 3

Liver of chlorpyrifos-treated chicken showing mononuclear cell infiltration around the blood vessels during 2nd month of treatment (H and E, ×400)

Renal changes were found to be mild up to 3rd week of the experiment. From 4th week onwards changes became prominent and were characterized by congestion, focal to diffuse hemorrhage, tubular degeneration, necrosis, and cellular swelling [Figure 4]. Glomeruli showed congestion and necrosis [Figure 5] with dilatation of Bowman's space and vacuolar degeneration.

Figure 4

Kidney of chlorpyrifos-treated chicken showing tubular degeneration, necrosis, and cellular swelling during 2nd month of treatment (H and E, ×400)

Figure 5

Kidney of chlorpyrifos-treated chicken showing glomerular congestion and necrosis during 3rd month of treatment (H and E, ×400)

Discussion

The increased Hb concentrations and TEC observed in the present study might be due to severe diarrhea causing dehydration resulting in hemoconcentration.[910] The gradual lymphopenia might be due to decrease production of lymphocyte in the lymphoid tissues resulting from the stress of intoxication.[11]

The significant heterophilia might be due to ingestion of CPF, which caused pathological stress in chickens due to noninflammatory disorders resulting in the rise of glucocorticoid level in circulatory blood.[9] Similar observations were recorded earlier in chickens due to CPF toxicity.[12]

The increased ALT, AST, and ALP values might be attributed to the liver damage in the toxicant fed birds. The results suggest that administration of CPF caused necrotic changes in the liver, as seen in histopathological study, thus causing leakage of the enzyme into the blood. Significant increase in AST and ALT was reported in goats fed with CPF.[13] AST is found in liver, skeletal muscle, heart, kidney, and brain in the variable amount between species. It is the last enzyme to rise after muscle or liver damage.[14] Increased levels of these enzymes have also been reported in the serum of birds in CPF toxicity.[15]

Reduced CHE activity is a reliable indicator of organophosphorus (OP) poisoning and a biomarker of absorption of OP insecticides.[16] Significant inhibition of CHE activity was reported in CPF intoxicated chicken.[15] CPF act through their active oxon metabolites and inhibits the target CHE. Plasma and other tissue CHE are important for assessing the extent of poisoning induced by organophosphates. Plasma CHE inhibition by 20–30% usually indicates exposure to organophosphate, whereas 50% inhibition or more is associated with serious poisoning and adverse effects.[17]

The fall in total protein could be due to the stress enhancing effect of CPF, or general toxic action that leads to decrease in weight in the treated birds.

The rapid increase in uric acid concentration might be due to acute renal disorders, as observed in histopathological study. The transient renal failure was due to both a direct action of the OP, causing tubular cell necrosis, and to a secondary mechanism that follows the cholinergic crisis, causing hypovolemic shock and rhabdomyolysis.[18] Mononuclear cell infiltrations in liver suggested onset of immunological response by the host. Lesions observed in liver in the present study are consistent with several workers.[19] Hepatotoxic effect of CPF reported in layer chickens[15] corroborate with the findings of the present study. Histopathological lesions of kidneys were indicative of nephrotoxicity of CPF and its metabolites as kidneys are the major route for elimination of CPF.[15] Since the renal tubules are particularly sensitive to toxic influences, in part because they have high oxygen consumption and vulnerable enzyme systems, and in part, because they have complicated transport mechanisms that may be used for transport of toxicants and may be damaged by such toxicants. The presence of necrosis may be related to depletion of ATP, which finally leads to death of the cells.[20]

Thus, it is concluded that exposure to CPF produces hematological, biochemical and pathological alterations in indigenous chicken. There is an obvious correlation between hematological, plasma biochemical, and histopathological lesions observed in the liver and kidneys of the CPF treated chickens. However, the exact mechanism that caused cell damage leading to hematological, biochemical, and pathological alterations needs to be elucidated.