Subchronic Acephate Exposure Induces Immunotoxicity in White Leghorn Cockerels

We investigated the subchronic immunotoxicity of the phosphoramidothioate organophosphorous insecticide, acephate in white leghorn cockerels (WLH). The cockerels were divided into ve groups; C1 (plain control), C2 (vehicle control), T1, T2, and T3 which received acephate suspended groundnut oil for 60 days at doses of 21.3, 28.4 and 42.6 mgkg −1 respectively. The live body weight gain, absolute and relative weights of the spleen, thymus, and bursa of Fabricius, hemoglobin (Hb), total erythrocyte counts (TEC), packed cell volume (PCV) and lymphocytes were signicantly decreased. However, monocytes, eosinophils, heterophils, and basophils were signicantly increased. Total protein, albumin and albumin to globin ratio, the antibody response to RD-F and delayed-type hypersensitivity response to DNCB dye or PHA-P, erythrocyte and brain Acetylcholinesterase activity was also signicantly reduced in T2 and T3. At 40 and 60 days of acephate exposure, nitrate and nitric oxide production by RD-F and mitogen Con A stimulated peripheral blood and splenic lymphocytes, as well as lymphocyte proliferation in response to antigen RD-F and mitogen Con A stimulation, were signicantly decreased in groups T2 and T3. Furthermore, dose-dependent increases in the frequency of micronuclei formation, varying intensity serum protein bands with different protein fractions (14.85KDa), and splenic DNA laddering (180 bp) were observed in groups T2 and T3. Histopathologically, the spleen and bursa showed morphological changes and mild lymphocyte depletion. In conclusion, low-level acephate exposure may affect acetylcholinesterase, lymphocytes, and immune responses in cockerels. As a result, it should be considered when assessing immunotoxicity and the risk to human and animal health. %depletion of lymphoids (mild variation of cortical lymphoid tissue); 3: 30–70% depletion of lymphoids (moderate variation of cortical tissue and follicle medular regions and increased stroma between follicles); 4:>70 % depletion of lymphoids (severe depletion of follicles with little to no cortex, increased stroma between follicles and decreased follicular size).


Introduction
Unregulated uses of pesticides often associated with modern agriculture can threaten the viability of the ecosystem by reducing biodiversity ( ora and fauna) and polluting natural resources, such as groundwater, which affect human health, communities and the environment [1,2]. Approximately ninety per cent of insecticides are not reaching to the intended species but signi cantly contaminate the ecosystem. Since the introduction of several pesticide regulation acts, there has been greater understanding of the gain and losses of insecticides use [3,4,5]. The issue of pesticide toxicity and other associated chemicals to non-target species continues to be a major concern worldwide [6]. dose(s) was based on previous studies [10] and the levels of acephate contamination / residue in drinking water or food supplies [11]. Acephate dissolved in peanut oil was injected directly into the crop through a catheter attached to 1 ml BD syringe. The cockerels were observed every day, and the body weights were reported at intervals of 7 days. At term, immune organs (Bursa of Fabricius, thymus, and spleen) were excised and weighed. All methods were carried out in accordance with relevant guidelines and regulations.

Body weight and relative lymphoid organ weights
The live body weight of birds was recorded prior to administration of acephate. Birds were observed daily and their live body weights were recorded at 7-day intervals for a period of 60 days in order to observe the manifestation of acephate. On day 61, after taking live body weight, ten cockerels of each experimental group were sacri ced and the spleen, thymus, and bursa of Fabricius were immediately removed and weighed separately for absolute organ weight. The relative body weights of the immune organs were determined by dividing the body weight of the organs. O: BW = Weight of the organ (g)/Body weight (g) X 10

Blood collection
Blood sampling under strict asepsis was done from cardiac puncture, jugular vein and wing vein of the cockerels of 0-10 day old (pre-experimental for checking maternal antibody titer), 10-40 day old and 40 day old, respectively, until the termination of experiment for hemato-immunological studies. At 20day intervals peripheral blood lymphocytes and serum were collected from heparinized (with 20 IU ml −1 heparin) and nonheparinized tubes, respectively. In immunotoxicity tests, peripheral blood lymphocytes were treated within an hour of collection, while serum was held at -80°C for further study.

Hematological studies
The day of the blood collection hematological tests were performed. The collected blood samples were diluted with 0.015 Toluidine blue and the total counts of leukocytes were assessed [12]. The differential leukocyte count was made in peripheral blood smears stained by Giemsa (Qualigens) [13].

Serum chemistry
To determine the non-speci c immune response in subchronic acephate-exposed cockerels serum total protein (gm dl −1 ), serum albumin (gm dl −1 ), and serum globulin (gm dl −1 ) were estimated with standard protocol of Core System as-say kits using an autoanalyzer (Photometer BT-224, Biotechnica Instruments, Italy) (Bt 1975 Jul).

SDS Polyacrylamide-Gel Electrophoresis (SDS-PAGE) of serum protein
The changes in the serum protein pro le were analysed by 12 % resolving gel prepared according to Laemmli (1970) containing 1.5 M Tris-HCl buffer (pH8.8), 10 % SDS, 10 % ammonium persulphate and TEMED along with a 5 %stacking gel. The stacking gel (5 %) was made using 1.5 M Tris-HCl (pH 6.8), 10 % SDS, 10 % ammonium persulphate and TEMED. The electrophoresis was accomplished at 150 V till dye came out of the gel using 1x Tris glycine buffer. The molecular weights of the serum protein subunits were determined using pre-stained molecular weight standards run along with the samples. The volumes of samples were adjusted to a concentration of 50μg proteins per well. On completion of electrophoresis, the gels were stained for proteins with 0.1 % Coomassie brilliant blue according to conventional methods and destained in a solution of 50 % methanol containing 10 % acetic acid. Prior to viewing, the gel was washed with distilled water until the background was clear. In reference to markers, the different protein fractions and molecular weights were estimated.
2.8 Acetylcholinesterase 2.8.1 Erythrocyte acetylcholinesterase We assessed depression of Acetylcholinesterase enzyme (AChE) activity, reported for organophosphate toxicity in red blood cells and brain by measuring acetylthiocholine iodide (ATChI) according to standard protocols [14]. AChE activity was determined in blood samples (10μL) collected at 20 day intervals and reported as μM ATChI hydrolyzed min −1 mg −1 protein.

Brain acetylcholinesterase
On day 61 all animals were sacri ced humanely by rapid decapitation. The brain was immediately removed from the head, and put in ice-cold saline. In order to measure brain acetylcholinesterase, dissection for discrete regions of the brain was performed on pre-chilled sterile Petri dishes. Great care has been taken to avoid tissue injury or in ammation of any portion of the brain when separating from the skull or dissecting the different areas [15]. The whole brains were suspended in a buffer (1 g of tissue plus 10 mL of sodium phosphate0.1 M, pH 7.5) and homogenised in an ice bath using a homogenizer. The homogenate was then centrifuged (5,000g/30 min/5°C), discarding the soluble portion. The pellet was then re-suspended using the equal volume of 0.1 g percent of Triton X-100 in 0.1 M sodium phosphate, pH 7.5, solution, and rehomogenized. The supernatant resulting from the resolubilization of the pellet was collected after a second centrifugation (15,000g/90 min/5°C). Brain AChE activity was spectrophotometrically tested in 0.1 M sodium phosphate, Triton X-100 0.1 g percent pH 7.5 by modifying Ellman's method [14]  In vivo, cell-mediated immune response was evaluated by assessing the DTH reaction to 2, 4-dinitro-1chlorobenzene (DNCB) dye by following an earlier standard method with minor modi cations [17]. Three cockerels from each group were arbitrarily selected and 2×2 cm 2 aseptically defeathered skin areas on the left and right abdomen of each cockerel were marked with India ink at day 50. 100μl DNCB (10 mg ml −1 ) dye dissolved in acetone and olive oil in 4: 1 ratio was applied to the marked areas of the right side and 100μl acetone (vehicle control) applied to the marked areas of the left side of the cockerel abdomen. The birds were challenged by applying 100μl DNCB (2 mg ml −1 ) dye at the same areas of right side and 100 μl acetone on the left side after 7 days of rst application. The increase in dermal thickness (mm) was measured at 0, 6, 12, 24, 48 and 72 h post-application with a vernier caliper. Increase in skin thickness (mm) = (T t −T 0 ) R− (T t −T 0 ) L Where T t = thickness at t, T 0 =thickness at 0 h, R = right side, L = left side and t = time. DTH reaction in the form of increase in dermal thickness was also recorded in phytohemagglutinin-P (PHA-P). To this end, on day 57, three birds were randomly selected from each group. 100 μl PHA-P (1mgml −1 ) dissolved in PBS had been injected into the third interdigital space of the right leg and 100 μl PBS (vehicle control) into the left leg. The increase in footweb thickness (mm) was measured at 0, 6, 12, 24, 48 and 72 h of post-application with a vernier caliper and foot web index was calculated. Foot web index = (T t −T 0 ) R− (T t −T 0 ) L Where T t =thickness at t, T 0 = thickness at 0 h, R = right side, L = left side and t = time 2.9.2 Anti-NDV antibody assay Cockerel humoral immunity was determined by assessing IgG antibody levels speci c to Newcastle Disease Virus (NDV) in serum samples using ND Ab Synbiotic ELISA kit (Hester Pharmaceuticals).
2.10 Lymphocyte mitogenesis assay 2.10.1 Separation of peripheral blood lymphocytes The peripheral blood lymphocytes were isolated according to previously re-ported standard protocols [18]. Brie y, 5 ml of the heparinized fresh blood from each cockerel was layered over 3 ml of histopaque-1077 in 15ml polypropylene centrifuge tube slowly through the wall and centrifuged at 1400 rpm for 40minutes.
Mononuclear blood lymphocytes circumscribed at the plasma and histopaque interface were collected in a separate centrifuge tube. The lymphocytes, after washing thrice with sterile PBS (pH 7.2), were suspended in1-2 ml of red-free phenol RPMI-1640 medium supplemented with 10 % bovine foetal serum, 25 mM HEPES, 24 mM NaHCO3, 2 mM l-glutamine, 100 IU penicillin ml −1 and 100 g of streptomycin ml −1 .

Separation of lymphocytes from spleen
The spleen was excised gently after the cockerels were sacri ced at the end of experiment on day 61, and splenocytes were isolated by following previously reported standard protocols [19,20]. The excised spleen was washed thrice in sterile ice-cold phosphate buffer saline (PBS). The spleen capsule was removed and the organ was sliced into sections with the forceps and the scissors in the PBS. With a 10 ml glass syringe, the fragments were gently pushed through the mesh into a Petri dish containing a chilled PBS. Cell suspension was collected in 15-ml centrifuge tubes and allowed to stand on the ice for 5 min to settle heavy tissue parts. The uppermost 12 ml of the cell suspension was collected and centrifuged for 10 min at 1400 rpm. Cell pellets were then resuspended in PBS and layered over histopaque-1077 at a ratio of 1:1. The tubes were centrifuged for 40 minutes at 1400 rpm. Further processing of the collected splenic lymphocytes was done as stated earlier to differentiate peripheral blood lymphocytes.

Cell viability count
The viable splenocytes was counted by using 0.1 % trypan blue dye, and hemocytometer [21]. The percentages of T and B lymphocytes in the peripheral blood and experimental bird spleen were determined using Annexin-V FITC Apoptosis Kit (Sigma Aldrich). The samples were analyzed by ow cytometry (BD FACS Aria, CA)

Lymphoproliferative response
In vitro the mitogen stimulation assay was performed as de ned in the literature with minor modi cations to evaluate lymphocyte proliferation [22].The active splenocytes reduced blue coloured 3-(-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) chemical dye to its purple colour insoluble formazan which was read at 492 nm by using 650 nm as a reference wavelength in ELISA reader (Multiskan EX, Thermo Lab Systems, Australia). The mitogen stimulation Index (SI) was determined as Stimulation Index (SI) = (OD s −OD us )/OD us Where, OD s and OD us indicate optical densities of stimulated cells and unstimulated cells, respectively.
2.11 Assessment of mononuclear cell functional activity 2.11.1 Nitrite production by mononuclear cells The functional activity of mononuclear cells stimulated by antigen Newcastle disease virus 'F' Strain (RD-F) or Lipopolysaccharides (LPS) was evaluated by assessing the concentration of nitrite produced by cultured cockerel splenocytes from control and treatment groups by following an established method [23]. The Nitrite level was assessed by measuring the absorbance at550nmwithin 30 minutes in the spectrophotometer micro plate reader (Multiskan EX, Thermo Lab systems, Australia). The concentration of nitrite produced by splenocytes was determined by comparison to the nitrite standard reference curve drawn with known sodium nitrite concentrations within cell-free medium and exhibited as μmol nitrite /3×10 6 cells.
Nitrite (μM) = (∆A550nm-intercept)/slope 2.11.2 Nitrate reduction by mononuclear cells (Phagocytic activity test) The nitroblue tetrazolium (NBT) dye reduction test was performed to mea-sure the phagocytic activity of splenic mononuclear cells following an established method [24]. The Nitrate level was assessed by measuring the absorbance at 710 nm immediately extraction of formazan using spectrophotometer (DU 640B, Beckman, USA). The concentration of nitrate reduction(formazan yield) by splenocytes was calculated as 1 nmol ml −1 formazan corresponds to an optical density 0.054+0.003 in both untreated control and acephate treated splenocyte.

Cytokinesis block Micronucleus assay in Cockerel splenic lymphocytes
The "in vivo biomonitoring" micronucleus (MN) test was performed to deter-mine the level of exposure and health risk, according to Krahn [25]. Brie y, cultured lymphocytes were taken and Colchicine (2×10 -7 nal) was added to each culture for the last 2 or 3 h of incubation. Metaphase cells were collected by "mitotic shake-off", sedimented by centrifugation, resuspended in 0.075 MKCl, and incubated at 37ºC for 5 min. The cells were sedimented and resuspended (three times) in methanol: acetic acid xative (3:1, v/v). Following a nal centrifugation and resuspension in a small volume of xative, the cells were dropped onto microscope slides. Slides were stained in 5 % Giemsa for 24 h, air dried, and mounted using permount. Air-dried and coded slides were analyzed using a light microscope. One thousand lymphocytes of each chick had been scored under oil immersion. Non-refractile particles, which in all respects resembled a nucleus, were considered to be micronucleus.

Internucleosomal DNA cleavage assay
In order to further evaluate the effect of acephate on apoptosis induction in splenic immune cells, an assay for internucleosomal DNA cleavage (a hall-mark of apoptosis) was carried out as previously reported [26]. Brie y, the fragmented DNA was extracted from splenocytes using a standard phenol: chloroform: isopropyl alcohol extraction (25:24:1), quanti ed by spectrophotometry, analyzed on a 0.8 % agarose gel, and photographed (digitized) (Gene Genius Bio Imaging System of SynGene, UK) and the electrophoretic pattern was utilized to determine total genomic DNA damage.

Histopathology
Bursa of Fabricius, spleen and thymus were xed in 10 % formalin -saline solution from each cockerel. After processing, para n-embedded sections of each tissue were cut at a thickness of 4-6μmand stained for histopathological examination using a microscope with hematoxylin and eosin (H and E) [27].In splenic tissues, the degree of lymphoid depletion was assessed by the extent to which white pulp was reduced in size and distribution, and lymphoid follicles were measured using a microscope (200x elds) at 20 different elds per portion. The histomorphological lesions were blindly evaluated by a veterinary pathologist and these assigned to numerical scores 0 to 4, where a higher value corresponds to increasing severity. The number or size of lymphoid tissue follicles or their germ centers was scored between 1 and 4 in bursal tissues. 1: 0-10 % depletion of lymphoids; 2: 10-30 %depletion of lymphoids (mild variation of cortical lymphoid tissue); 3: 30-70% depletion of lymphoids (moderate variation of cortical tissue and follicle medular regions and increased stroma between follicles); 4:>70 % depletion of lymphoids (severe depletion of follicles with little to no cortex, increased stroma between follicles and decreased follicular size).

Statistical analysis
Data were analyzed one-way analysis of variance (ANOVA) using SPSS 17.0software with Tukey's HSD posthoc tests to determine whether there were any statistically signi cant differences among experimental groups.
The results are given as mean ± standard error mean (X±SEM). The p≤0.5 was deemed signi cant differences.
All methods are reported in accordance with ARRIVE guidelines as dose treatments were given.

General assessment
An abrupt depression, decreased feed intake, dullness and ru ed feathers were shown in cockerels exposed to extremely toxic (T3-42.6 mg kg −1 ) doses of acephate over 60 days. While in medium (T2-28.4 mg kg −1 ) and low (T1-21.3 mg kg −1 ) toxic dose groups clinical signs were milder in appearance. The cockerels from control groups (C1 and C2) did not exhibit any abnormal behavior and symptoms of toxicity. Birds died in treatment groups with serious nervine symptoms and clinical signs of toxicity before death. In the eighth week of experiment, mortality reached six, four and two in group T3 (21. Mg kg −1 ), groupT2 (28.4 mg kg −1 ) and group T1 (42.6 mg kg −1 ) respectively. The remaining birds of all treatment groups revealed characteristic signs of organophosphate toxicity.

Body weight and lymphoid organ weights
Supplementary FIGURE 1 presents the stress induced toxicological impact of oral administration of acephate over 60 days on the live body weight of cockerels. Acephate exposure altered the feed intake in the cockerels of an extremely toxic dose group (data not shown) that led to signi cant decrease in live body weight of cockerels weighed at 7-day intervals in dose dependent manner compared to controls. TABLE 1 illustrates the effects of acephate exposure in cockerels upto 60 days on absolute organ weights and relative weights of cockerels. Absolute and the relative weights of immune organs viz spleen, bursa of Fabricius, and thymus, were signi cantly decreased dose dependently in medium and extremely toxic dose groups (T2 and T3) in comparison to controls except for the low dose treatment group (T1). Table 2 and 3 illustrate the impact of subchronic exposure to acephate on hematological indices, peripheral blood lymphocytes, total leukocyte counts and differential leukocyte counts (DLC) in WLH cockerel from treatment and control groups. Blood testing of cockerels exposed to acephate per os for 20 days did not show any signi cant alterations in Hb, TEC, and PCV whereas these were signi cantly reduced in cockerels from treatment groups (T2 andT3) on days 40 and 60 (TABLE 2). TLC (FIGURE 2) and lymphocyte concentrations were also signi cantly reduced dose dependently on days 20, 40, and 60. On the contrary, there was a dose dependent rise in the number of heterophils, monocytes, eosinophils, and basophils in the cockerels from treatments groups in comparison to controls (TABLE 3).

Total protein
Acephate exposure for 20, 40, and 60 days caused the signi cant reduction in the serum total protein level in the cockerels from medium (T2) and extremely toxic (T3) treatment groups compared with controls (FIGURE 3A).

Serum albumin
Acephate exposure for 20, 40, and 60 days caused the signi cant reduction in the serum serum albumin level in the cockerels from medium (T2) and extremely toxic (T3) treatment groups compared with controls (FIGURE 3B).

Serum globulin
Acephate exposure in cockerels with medium toxic (T2) and extremely toxic(T3) dose(s) caused signi cant decrease in serum globulin level at day 20while it was non signi cantly diminished in all treatment groups at day 40and signi cantly decreased in T2 and T3 at day 60 compared with controls (FIGURE 3C).

Serum Albumin: Globulin ratio
Serum albumin showed a signi cant decline in albumin: globulin ratio at medium and extreme toxic dose levels compared with controls (FIGURE 3D).

Changes in serum protein pro le
Electrophoretic analyses of chicken serum in the treatment groups re ected the appearance of varying intensity protein bands compared to controls. Nine molecular entities ranging from 16.42-175 kDa molecular weight were detected in chicken serum at a low toxic dose (21.3 mg kg −1 ) of acephate and control groups. in the serum protein of Acephate exposed cockerels at an extremely toxic dose (42.6 mg kg −1 ) of up to 60 days (FIGURE 4). This was con rmed by the reduction of total proteins as shown in the Figure 3A. The expression of all fractions had become too weak to be detected in all test groups.

Erythrocyte acetylcholinesterase
In vivo effects of acephate on erythrocyte acetylcholinesterase activity are presented in TABLE 4. Acephate exposure in white leghorn cockerels caused a signi cant decrease in erythrocyte acetylcholinesterase activity in a dose-dependent manner as compared to control groups. The maximal inhibition on the acetylcholinesterase was observed 20 days onwards which was in a dose-dependent manner.

Brain acetylcholinesterase
In vivo effects of acephate on brain acetylcholinesterase activity are shown in TABLE 4. Acephate exposure in white leghorn cockerels caused signi cant decrease in acetylcholinesterase activity as compared to control groups.
3.6 Assessment of cell mediated and humoral immune response 3.6.1 Delayed type hypersensitivity (DTH) Cell mediated immunity was assessed through measuring DTH reaction using DNCB dye in response to the acephate exposure at varying toxic doses to cockerels over 60 days. The mean rise of dermal thickness (mm) was seen in all treatment groups at 6, 12, 24, 48, and 72 h after pre challenge and 7-days' later postchallenge sensitization with DNCB is shown in FIGURES 5A-5B. The rise in dermal thickness after sensitization with DNCB dye was signi cantly decreased in medium (T2) and extremely (T3) toxic dose groups at 12, 24, 48,and 72 h in dose dependent manner compared with the controls. However, anon-signi cant increase in dermal thickness was also recorded in a low toxic dose (T1) treatment group at 12 and 24 h (FIGURE 5A).
However, the rise in dermal thickness of cockerels following post-challenge sensitization after 7-days of prechallenge sensitization was signi cantly reduced at 12, 24, 48 and72 h in medium (T2) and extremely (T3) toxic dose groups comparison with control groups, and this impact was also evident as a non-signi cant increase in dermal thickness in cockerels exposed to low toxic dose (FIGURE 5B). DTH response was also evaluated, followed by interdigital PHA-P injection and measurement of footweb index (mm) at 6, 12, 24, 48 and 72 h post-injection. Acephate decreased the PHA-P post-injection footweb index at 12, 24, 48, and72 h (FIGURE 5C).

Assessment of humoral immune response
The immunological reaction in response to the antigen challenge is the best way to evaluate the immune system. That approach has therefore been applied in this analysis. Humoral immunity in response to RD-F antigen in cockerels exposed to acephate over 60 days was recorded by assessing the level of anti NDV antibody titer using ELISA and shown in FIGURE 6. On the 20th day of acephate exposure anti NDV ELISA values were non-signi cantly higher in cockerels from low toxic dose group (T1) and signi cantly lower in medium toxic (T2) and extremely toxic (T3) dose groups. While on days 40 and 60 after the start of exposure to acephate, these values were signi cantly lower in all treatment groups in dose dependent manner compared with controls and this decreasing effect on antibody production persisted till the end of study (FIGURE 6).

Cell viability
The viability of splenocytes was found to be around 70 per cent at the treated concentrations. Viable cells appear translucent, and dead cells stained blue due to increased permeability of the cells.

Splenic and peripheral blood lymphocyte proliferation
The effects of acephate exposure over 60 days on immune responses of cockerels determined by assessing in vitro proliferation of splenic or peripheral blood lymphocyte stimulated with antigen RD-F or mitogen Con A.
Results of lymphocytes' stimulation index are shown in FIGURE7A, 7B, and 7C. The toxic effect of acephate on peripheral blood lymphoproliferation after stimulation with antigen or mitogen was evidently signi cant from day 40 onwards to the end of study (FIGURE 7A and 7B). On Day20, lymphoproliferation was signi cantly increased in cockerels from the low toxic dose (T1) group, while it was signi cantly decreased in cockerels exposed to medium and highly toxic acephate levels compared to cockerels from the control group. Signi cant reductions in lymphoproliferation were observed in cockerels from medium (T2) and extremely (T3) toxic dose groups compared to cockerels from control groups from day 40 to the end of the study. How-ever, the lymphocyte proliferation of splenocytes stimulated with antigen and mitogen was signi cantly reduced in all treatment groups dose dependently, and the effect was more marked in the birds subjected to extremely toxic dose(T3) (FIGURE 7C).
3.8 Functional activity evaluation of mononuclear cells 3.8.1 Assessment of non-speci c immune response (nitrite production) The concentrations of nitrite were determined to assess the production of nitric oxide (NO) by peripheral blood and mononuclear splenocytes. Acephate induced development of nitrite by peripheral blood lymphocytes stimulated with antigen LPS and RD-F or mononuclear splenocytes of cockerels stimulated with mitogen Con-A is shown in FIGUREs 8A, 8B, and 8C respectively. Acephate exposure over 20 days resulted in signi cant reduction in the production of nitrite by peripheral blood lymphocytes stimulated with LPS and RD-F in the birds from medium (T2) and extremely (T3) toxic dose groups as compared to controls. However, the production of nitrite by peripheral blood lymphocytes was signi cantly reduced in birds of all toxic dose groups (stimulated with LPS) and dose-dependent manner (stimulated with RD-F) compared to controls at day 40, and at day 60, signi cantly decreased in dose-dependent manner (stimulated with LPS) and in birds of all toxic dose groups (stimulated with RD-F) compared to controls (FIGUREs 8A and 8B). The similar dose dependent signi cant reductions in nitrite production by mononuclear splenocytes stimulated with mitogen Con-A were also recorded (FIGURE 8C).

Assessment of Macrophage Function test (nitrate production)
The synthesis of nitrate (formazan) in 1 mL supernatant of harvested splenocytes was used to assess phagocytic activity of splenic mononuclear cells. FIGURE 9 shows nitrate production by splenic mononuclear cells (stimulated with ConA) in birds exposed to Acephate. The results indicate that treatment with Acephate for 60 days reduced nitrate production by ConA stimulated splenic mononuclear cells in a dose-dependent manner, with a non-signi cant increase in nitrate production by low toxic dose (21.3 mg/kg) exposed birds at 24 h, and that this signi cant change persisted until the end of the study (FIGURE 9).

Cytokinesis block micronucleus (CBMN-Cyt) assay
Acephate increased the development of micronucleus, nuclear buds and nucleoplasmic bridges in a concentration-dependent manner in the cytokinesis block micronucleus cytome (CBMN) test when compared to the control. At higher concentrations, however, these increases were evident. The internucleosomal assay revealed that acephate signi cantly increased DNA damage. In addition to the induction of micronuclei (TABLE 5 and FIGURE 10A-D), the acephate also caused other nuclear anomalies in the treated sequence, such as vacuolated nuclei and binucleated cells with unequal nuclear content. In certain cases, chromatin disintegration and karyorrhexis were also observed.

Molecular detection of apoptosis by DNA ladder assay
The results showed the formation of DNA ladders in all splenic cells treated with acephate that indicate apoptotic cell death (FIGURE 11). The splenocytes of cockerel exposed to acephate triggered intense laddering and increased smearing, which suggests spontaneous cleavage of DNA and necrotic cell death. Splenic cells from control cockerels have a vivid top lane band with intact DNA and less evident apoptotic laddering.

Histopathology
Spleen and bursa of Fabricius histopathology exams showed changes primarily in group T3 (42.6 mg kg −1 body weight). Severe depletion of lymphocytes, necrotic foci and red pulp expansion was observed in the spleen (FIGURE 12A-D), whereas there was no histopathological lesion in the thymus (data not shown).
Bursa of Fabricius showed lymphoid depletion and decreased follicular size with inter follicular brosis (FIGURE 12E-H). These histopathological severities were in a dose-dependent manner and the scorings are depicted in TABLE 6.

Discussion
The continuous usage of pesticides pollutes the environment and cause a detrimental effect on the health system, Half-life of acephate dissipation in aerobicand anaerobic soils is less than 3 and 6 days, respectively, and degrades to im-mobile compounds in 20 days [28]. The toxic doses of acephate were selected based on the basis of residues and predicted concentrations reaching non-target species through feed and water after one to two consecutive half-lives. In this context, we hypothesized that avian species, due to having an impor-tant immune organ bursa of Fabricius, may act as an indicator for detecting environmental contaminants present in low concentrations. Often it has been observed that environmental toxicants present at low concentrations affect immune organs, which leads to immunosuppression. Here, we chose cockerel asthe most appropriate and widely used experimental model to investigate the immunotoxic effects of acephate. We recorded a substantial decrease in cockerel live body weights as well as the weights of immune organs such as thethymus, spleen, and Fabricius bursa, and these ndings were corroborated by other studies in broiler chickens [29]. The weights of the spleen, thymus, and bursa of Fabricius were found to be associated with the dosage and length of acephate exposure, as well as the relative immune status of the animals. Our results indicate a dose-dependent relationship between reduced organ weights and humoral and cell-mediated immunity in cockerels from treatment groups relative to controls after 8 weeks of exposure.
Blood provides a signi cant pro le for studying the toxicological effect on animal tissues. In current exploration, acephate administration negatively affects Hb, TEC, PCV, and TLC, these changes correlated with dose and duration of exposure [30]. This shows the target action of acephate on erythropoiesis, which eventually results in hemolysis and anemic conditions of cockerel. White blood cells (WBCs), also called leukocytes, are part of the body's immune system, which helps to ght infection and other diseases. In cockerels, there are ve major kinds of leukocytes (heterophiles, eosinophils, basophils, lymphocytes, and monocytes), each responding differently to protect against foreign organisms. We have analyzed the reduced level of TLC (leukopenia) in the acephate exposure cockerels, and this shows hematological dysfunctions and immune de ciency. Our previous study has shown a similar nding (leukopoiesis suppression) after short term exposure (28 days) of acephate [10] and also this results in agreement with ndings of imidacloprid exposure in male WLH cockerels [31]. On the contrary, we observed signi cantly increased heterophile, monocytes, eosinophil and basophil percentage in blood smear of acephate treatment groups. The most common and important cause of heterophilia is a systemic infection and these ndings are corroborated with histopathological changes of kidney viz., intertubular haemorrhages, tubular degeneration, tubular dilatation and fatty alterations (data not shown). Although the exact mechanism for this effect is not yet wellknown but earlier investigators have reported that serine hydroxylases associated with cell membranes of lymphocytes and monocytes altered in such a way that may induce structural/functional changes in immune cells including lymphoproliferation in response to antigens [32]. The albumin globulin ratio and total serum proteins can provide useful information about the function of the liver and lymphocytes. Our ndings show that after acephate intoxication, total protein and the albumin/globulin ratio both drop signi cantly. The amounts of globulin, on the other hand, were insigni cant. As a result of its stressogenic effects, acephate can impair protein metabolism and liver functions, affecting cockerel weight gain (Supplementary FIGURE 1), and these are typical insecticide side effects. As compared to controls, the homogenates of Acephate exposed and non-exposed chicken serum re ected the presence of varying intensity protein bands in a dose dependent manner. Following 60 days of exposure to various concentrations of Acephate in developing WLH cockerels, the eletrophoretic pattern of chicken serum protein revealed clear differences in band numbers and molecular weights. These ndings support previous research that showed the appearance of a new protein band with a molecular weight of 74 KDa after 15 days of cyanophos insecticide treatment of Albino rats [33]; pyriproxyfen, Bioneem, Garlic Gard, Nat-1, and deltamthrin caused an increase in the amount of bands in separated soluble proteins of Spodoptera littoralis eggs [34]; Glyphosate and CdCl2 displayed marked differences in serum enzyme activity and polyacrylamide gel electrophoretic patterns of serum proteins in an in vitro sample, but not paraquat [35]. The appearance of a new protein band, similar to the cyanophos pesticide in human serum protein, could be due to the Acephate's a nity for either high molecular weight lipoprotein or binding to transferrin or albumin [36]. Our ndings indicate a strong link between inhibition of the activity of the brain and erythrocyte AChE and the dose of acephate. These ndings are in agreement with other reports [37]. Also, this result is new and there are limited reports on AchE inhibition activity after subchronic exposure of acephate. Reduction of cholinesterase is common in all organophosphate pesticides in both the blood and tissues. Cholinesterase inhibition causes the accumulation of acetylcholine in synapses, resulting in nervous system (neurobehavioral activity) and gastrointestinal tract (hypophagic activity) dysfunctions [38] [39]. The DTH and foot web index assay in response to DNCB dye and PHA-P, respectively, is of great usage for assessing the overall competency of cell-mediated immunity within the host and is an in ammatory response mediated by T-cells. Subchronic acephate exposure suppressed the footweb index at 12, 24,48 and 72 h post-injection PHA-P. The ndings indicate that exposure to acephate at moderate and high toxic dose rate induced dysfunction of another T-cell subpopulation (T effector cells) and toxic to cell-mediated immune response. These cells produce various cytokines following interaction with a particular antigen that plays a crucial role in DTH. In the DTH reaction, the primary lymphocyte response appeared to be responsible for the accretion of mononuclear cell in ltrates, interaction and increased vascular permeability that occurs in the vicinity of a stimulus [6]. This nding gets further support as we reported a reduced number of total leukocyte counts and absolute counts of lymphocytes in groups treated with acephate. We recorded signi cantly in-creased levels of antibodies against ND vaccine (RD-F), which were contained in serum obtained from low-dose groups of cockerels. However on days 40 and60 of the experiment, these were signi cantly lower in subsistence treatment groups. The decline in vaccine response suggests that subchronic acephate exposure has impaired the production of B-cell antibodies and memory response [40]. This nding led to an obvious immunostimulation and subsequent immunosuppression at minimal and higher acephate concentrations. The exact mechanism of this immunosuppression has not been de ned. The subchronic acephate exposures resulted in a signi cant reduction in peripheral blood lymphocyte and mononuclear splenic lymphocyte proliferation in response to anti-gen LPS and RD-F and mitogen Con A. The effect was noticeable after 20days of exposure in all treatment groups and persisted until the end of the study except for a signi cant increase in the low toxic dose treatment group at day 20 [41]. A signi cant decrease in B-lymphocyte proliferation suggests the decreased capacity of B-lymphocytes to form clones and turn them into plasma cells.
Plasma cells are responsible for the secretion of antibodies, suggesting that B-lymphocytes have become less receptive to antigen, leading to suppression of humoral response. The impaired humoral response could increase the risk for vaccine failure and susceptibility to infection. The decrease in cockerel lymphocytes proliferation in response to antigen LPS and RD-F and mitogenic Con-A may be indirectly correlated with the decrease in proin ammatory cytokines, lymphocyte enumeration, and differentiation. However, the mechanisms involved in the above-mentioned effects are not well under-stood. All the treatments in this study signi cantly suppressed nitrite production by peripheral blood and splenic mononuclear lymphocytes stimulated with antigen (RD-F) and LPS and mitogen Con A. Supporting our results, ithas been reported that rats exposed to a low level of acephate inhibited iNOS production which was also similar to iNOS production by splenic mononuclear splenocytes stimulated with LPS [42]. It has been indicated that a suppressive effect on NO production could take place at different molecular levels [43].Phagocytic cells are considered to be an important component of the defense mechanisms, since they act against any foreign invasion not only to kill and remove them from the body, but they also act as antigens presenting cells and actively participate in the speci c immunity. In this study, all treatments signi cantly suppressed the generation of nitrate by splenic mononuclear cells stimulated by the mitogen Con-A, except for a non-signi cant increase in cockerels in the low-toxic dose group at 12 h of stimulation. Similar to the present observations on the effect of acephate on lymphocyte activity, signi cant depression in the functional activity of macrophages as assessed by the nitroblue tetrazolium reduction test has been reported in broiler chickens fed a diet carbaryl at 20 ppm [44]. Contrary to the present ndings in chickens, the NBT test showed a decrease in the number of active phagocytic cells in spleen at0.1μg/egg and 0.05μg/egg doses of combination insecticide treatment, while0.01μg/egg did not show any changes [45]. Decrease in lymphocyte nitrate production (NBT) indicates the reduced functional status of phagocytic cells. Impaired phagocytosis may also be due to the adversely modulated immunogenic potency of lymphocytes. Reduction in the number of active phagocytic cells in Acephate treated birds may also lead to a decrease in natural resistance or innate immunity to infections. The micronucleus test is one of the most reliable cytogenetic bioassays to detect mutagenic environmental agents DNA ladder assays were performed to determine apoptotic or necrotic type of speci c cell death which resulted in cleaving of cockerel splenic DNA into 180 bp as a ladder pattern from treatment groups in comparison to the control groups. The presence of the DNA ladder in splenocytes con rmed the acephate-induced apoptotic death. DNA cleavage has been suggested as one of the rst irreversible and characteristic phenomena that trigger apoptosis in fully grown immune cells [10]. The degree of DNA smearing is indicative of necrotic cellular events, including spontaneously splitted DNA. In the course of time the intensity of smearing increased. We carried out histopathological evaluations of lymphoid organs, spleen, thymus, and bursa of Fabricius to assess acephate induced im-munotoxic effect. The results showed that necrotic cells were increased in the splenic corpuscles dose dependently, which indicates that excessive necrosis in the spleen would eventually compromise the B and T cell based immune response. Severe lymphoid depletion of follicles with reduced follicular size and increased stromal follicle means variations in cortical lymphoid and development of inter follicular brosis. Similarly, lymphocyte depletions in focal areas of spleen and bursa of Fabricius have been observed in broiler chickens [49]. Acephate's immunotoxic effect is consistent with our previous research on other organophosphate pesticides. It has been shown that acephate inhibits both humoral and cellular immunity among chickens [10]. There is little in-formation about the way acephate pesticides act on the immune system. We suggest that inhibition of AChE can be the hierarchical event that contributes to the production of immunotoxicity in cockerels exposed to acephate (Supplementary FIGURE 2).
Immunosuppression in cockerels may result from direct action of acetylcholine on the immune system, and/or may be due to the toxic chemical stress related cholinergic poisoning. This is unlikely to require a direct cholinergic process, since the introduction of multiple cholinergic agonists will not inhibit immune reactions in vitro. Carbamates and organophosphate have been reported to work through serine esterase inhibition to in ect the generation and manifestation of an immune response [42].

Conclusion
In conclusion, subchronic exposure to acephate at concentrations relevant to the environment signi cantly altered the immunological functions tested in cockerels and could pose a signi cant risk of pathogen infection.
Our experimental ndings, in summary, endorse acephate's immunotoxic nature. Cellular immunosuppression can be measured almost precisely only by several parameters. Therefore, before concluding, it is necessary to analyze multiple parameters, as set out in this report (  Tables   TABLE 1: Trends of absolute and relative weights of immune organs in white leghorn cockerels upon daily exposure to acephate dissolved in peanut oil over 60 days. Results (mean ± SEM, n = 10) of absolute organ weight (g) and relative weight in the same column bearing (*) decreased signi cantly (p < 0.05), (**) strongly signi cantly (p≤0.01) and (***) profoundly signi cantly (p≤0.001) in Duncan's multiple comparison post hoc test.        Immunotoxicity induced by subchronic exposure to acephate in avian experimental model. The current study presents severe splenic and bursal lymphoid depletion and increased bursal follicle stroma, DNA laddering, reduced Nitrite and nitrate production by peripheral blood lymphocytes stimulated antigen RD-F and LPS and splenic mononuclear cells stimulated with mitogen ConA and reduced humoral immunity in all treatment groups on days 40 and 60 after the start of exposure to acephate compared with the controls.

Figure 2
Trends of Total Leukocyte Count (TLC) in cockerels upon daily exposure to acephate dissolved in peanut oil over 60 days. Each bar is a mean ± SEM of 10 cockerels. Bars with different alphabets display the mean difference at level 0.05 while bars with bullet point (•) and asterisk (*) vary signi cantly ( p < 0.05) and strongly signi cantly (p < 0.01) in Tukey's HSD post-hoc tests, respectively.   signi cant for the level 0.05 whereas bars with bullet point (•) and asterisk (*) vary signi cantly (p < 0.05) and strongly signi cantly (p < 0.01) respectively in Tukey's HSD post-hoc tests.

Figure 6
Trends of antibody response in white leghorn cockerels to RD-F following repeated acephate exposure dissolved in peanut oil upto 6o days. Each bar re ects the mean ± SEM of ELISA values (n = 10). Bars with distinctive letter set display the mean difference is signi cant at the level 0.05 while bars with bullet point (•) and asterisk (*) vary signi cantly (p < 0.05) and strongly signi cantly (p < 0.01) respectively in Tukey's HSD post-hoc tests. (•) and asterisk (*) vary signi cantly (p < 0.05) and strongly signi cantly (p < 0.01) respectively in Tukey's HSD post-hoc tests. Page 32/36 exposed to acephate dissolved in peanut oil over 60 days after stimulation with antigen RD-F and LPS. Bars with distinctive letter set display the mean difference is signi cant at the level 0.05 while bars with bullet point (•) and asterisk (*) vary signi cantly (p < 0.05) and strongly signi cantly (p < 0.01) respectively in Tukey's HSD post-hoc tests.

Figure 9
Nitrate production by splenic mononuclear cells of white leghorn cockerels following repeated acephate exposure dissolved in peanut oil over 60 days, after stimulation with mitogen ConA. Bars with distinctive letter set display the mean difference is signi cant at the level 0.05 while bars with asterisk (*) vary signi cantly (p < 0.05) and (**) strongly signi cantly (p < 0.01) respectively in Tukey's HSD post-hoc tests. Figure 10 (A-D) Trends of frequency micronuclei formation in cultured splenic lymphocytes of white leghorn cockerels upon daily exposure to acephate dissolved in peanut oil over 60 days. Each bar re ects the mean ± SEM of frequency micronuclei (n = 10). Bars with distinctive letter set display the mean difference is signi cant at the level 0.05 while bars with bullet point (•) and asterisk (*) vary signi cantly (p < 0.05) and strongly signi cantly (p < 0.01) respectively in Tukey's HSD post-hoc tests.  Supplementary Files