Iraqi Journal of Veterinary Sciences

Using T cell lymphokines to enhance the immune response against Newcastle disease in vaccinated broiler chickens fed naturally contaminated diet with different mycotoxins

B.H. Saud¹ and M.T. AL-Zuhariy^2*

¹Department of Pathology and Poultry Diseases, College of Veterinary Medicine, ²Department of Pathology and Poultry Diseases, College of Veterinary Medicine University of Baghdad, Baghdad, Iraq, ^* Email: dmshtak27@gmail.com

Abstract

The current study aimed to reduce the toxic effect of different mycotoxins and enhance the immunity against ND virus in broiler chickens by using lymphokines from hyperimmunized birds with Salmonella typhymurium. The study included three stages, the first stage included isolating Salmonella typhymurium. The second stage was immunized chicks with Salmonella typhymurium. The final stage of the study was accomplished by treating 250 broiler chicks (divided into 5 groups, 50 chicks /each) with the following treatments; G1: 0.5 ml lymphokines was injected I/P at day one old with live ND vaccine (la Sota strain) after 30 minutes in drinking water, the process repeated after 10 days; G2: the same as in G1 but inactivated killed vaccine was used s/c,no repetition was carried out at 10 days ; G3: a combination of G1 and G2 with revaccination of live La Sota vaccines only at 10 days; G4 : only vaccinated with live La Sota vaccine repeated at 10 days; G5: no treatments (negative control). All groups were challenge with local isolate of NDV (100ELD₅₀ 10⁵) at 25 days, all groups except the fifth group were fed on contaminated diet withmycotoxin. The results of the present study showed a significant increase (P <0.05) in antibodies titre against ND in the third group, followed by the first and second groups Measured by ELISA and hemagglutination (HI) test, A significant decrease (P >0.05) in the oxidation status (H₂O₂, MDA and LPO) and significant increase in the antioxidant defense (GSH-PX) in the liver and spleen samples. We conclude from the current study that the Salmonella immune lymphokines (SIL) helps in enhancement the level of immunity against Newcastle disease and n reduction the side effects of which mycotoxin.

Key words: ND, SIL, Mycotoxin, T cell, ELSA, HI

DOI: 10.33899/ijvs.2019.125977.1204, © 2020, College of Veterinary Medicine, University of Mosul.

This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).

استعمال اللمفوکاینز للخلایا التائیة لتعزیز الاستجابة المناعیة ضد مرض النیوکاسل فی دجاج اللاحم الملقح المستهلکة لعلائق ملوثة طبیعیا بسموم فطریة

بشیر حمید سعود و مشتاق طالب الزهیری

فرع الأمراض وأمراض الدواجن، کلیة الطب البیطری، جامعة بغداد، بغداد، العراق

الخلاصة

هدفت الدراسة الحالیة الى تقلیل التأثیرات السمیة للسموم الفطریة وتعزیز المناعة ضد مرض النیوکاسل فی الدجاج اللاحم باستخدام اللمفوکاین من الطیور الممنعة بثلاث جرع من Salmonella typhymurium. تضمنت الدراسة ثلاث مراحل، المرحلة الأولى شملت عزل Salmonella typhymurium، أما المرحلة الثانیة تضمنت تمنیع الافراخ بالسالمونیلا تایفیموریم، أما المرحلة الثالثة والأخیرة من الدراسة تضمنت اخذ 250 فرخة اللاحم بعمر یوم واحد، وقسمت الى خمس مجامیع کل مجوعة 50 فرخة، حقنت المجموعة الأولى بعمر یوم واحد 0.5 مل بالتجویف البریتونی Lymphokines وبعد 30 دقیقة لقحت بلقاح النیوکاسل (La Sota strain) عن طریق ماء الشرب، واعیدت الحقن واللقاح بعمر 10 یوم. اما المجوعة الثانیة حقنت بعمر یوم واحد 0.5 مل بالتجویف البریتونی Lymphokines وبعد 30 دقیقة لقحت فقط باللقاح الزیتی للنیوکاسل (La Sota strain) حقن تحت الجلد بعمر یوم واحد. وحقنت المجموعة الثالثة بعمر یوم واحد 0.5 مل بالتجویف البریتونی Lymphokines وبعد 30 دقیقة لقحت المجوعة الثالثة باللقاحین لمرض النیوکاسل بنفس العترة (الزیتی تحت الجلد واللقاح الحی عن طریق ماء الشرب بعمر یوم واحد) وأعید التلقیح فقط باللقاح الحی بعمر 10 مسبوق Lymphokines. أما المجموعة الرابعة لقحت بلقاح النیوکاسل (La Sota strain) عن طریق ماء الشرب (1 و 10) یوم ولم تحقن Lymphokines واعتبرت مجموعة سیطرة موجبة. أما المجموعة الخامسة لم تلقح ولم تحقن Lymphokines واعتبرت مجموعة سیطرة سالبة. جرعة عن طریق الفم جمیع المجامیع بالعزلة المحلیة لمرض النیوکاسل بجرعة (100ELD₅₀ 10⁵) بعمر 25 یوم، کل المجامیع غذیت على علیقة ملوثة بسموم الفطریة ما عدا المجموعة الخامسة. أظهرت نتائج الدراسة الحالیة وجود زیادة معنویة بمستوى (P<0.05) بمعیار الأجسام المضادة لمرض النیوکاسل فی المجوعة الثالثة تلتها المجموعة الأولى ومن ثم الثانیة بفحص الالیزا وفحص أثباط التلازن الدموی، وانخفاض معنوی بمستوى (P<0.05) فی معدلات إنزیمات الحالة التأکسدیة (H2O2، MDA and LPO) وارتفاع معنوی بأنزیم المضاد للتأکسد (GSH-PX) فی الکبد والطحال،. نستنتج من الدراسة الحالیة أن استخدام (Salmonella immune lymphokines SIL) یساعد على تعزیز المناعة اللقاحیة ضد مرض النیوکاسل وتقلیل التأثیرات الجانبیة للسموم الفطریة.

Introduction

Mycotoxins are diverse group of harmful particles to animal and human, which are the secondary metabolites of different toxigenic molds, especially aspergillus, penicillium and fusarium species, mycotoxins are produced in the grains as well as in the pre- and post-harvest feed in different environmental conditions and due to the large diversity of their toxic effects and their synergistic properties therefore, mycotoxins consider more risky to the consumers of food and contaminated feed (1). The consumption of low concentrations of ycotoxins for long periods exposes cells of the intestinal mucosa to damage, however the intestinal mucosa possesses both natural and specific immune components (2). Bouhet and Oswald (3) the main function of physical barrier of the intestinal mucosa is the transfer the electrical resistance through the epithelial layer, which found in the monolayer cells. Mclaughlin et al. (4) show that mycotoxins damage the proteins found in cell junctions and thus reduce the transmission of electrical impulses which is the transfer of cytokines between immune cells. Metabolic products of innate toxins affect the immune system of the intestinal mucous, which in turn significantly affects the performance and productivity of the animal, because the stimulation of mucous immunity is important in protecting against many of the germs that invade these surfaces and enter the body and cause diseases (5). It is known that oral vaccination is one of the most practical and economical methods in poultry However, the presence of mycotoxins in diet interferes with the immune response by affecting the mucous membranes and thus reduces the level of local immunity resulted after vaccination, making birds vulnerable to infectious and rapidly spreading diseases (6). Newcastle disease is a contagious, endemic and rapidly spreading disease that causes significant economic losses to the poultry industry. The pathogen of this disease is Paramyxovirus, Genus Avulavirus, family Paramyxoviridiae (7). Although many vaccine programs are used to ND control, the disease is endemic in many countries (8). Some research has confirmed that immune response is enhanced by many herbal and vitamins, but immune response is still low due to mycotoxins (9). Rashad and Mushtaq (10) confirmed that the use of lymphokines from Salmonella Typhmurium (Slmonella immune lymphokines SILK), enhanced the immune response against the ND challenge without using the vaccine by stimulating many immune cytokines, which are regulatory proteins produced by some Immune cells in to protect the body against infections. Mushtaq et al. (11) confirm use of SILK in the early days stimulated the immune response to protect the chicks from infectious bronchitis disease after the challenge of local isolation of the disease. Mushtaq, (12) also showed the role of SILk in promoting the maternal immunity at one day-old against avian influenza infection. Therefore, we expect that the addition of SILK to mycotoxins -contaminated diets could reduce the effects of mycotoxin during oxidative reduction and increased antioxidant defense systems with high response against ND.

Materials and methods

Detection of Mycotoxin in diet

Mycotoxin was detected using the ELISA test. A 20-gram feed sample is mixed with methanol (Fisher, Pittsburgh, PA, USA). Mixed with water (30/70 vlv) and shaking for 3 minutes. Then the mixture filtrated with Whatman filter (Whatman Clifton, NJ, USA), collects the supernatant and determined the concentration of toxins using ELISA kit (Agra Quantum mycotoxin assay, Romer, Singapore). Starter and finisher diet (A1) for (G1, G2, G3 and G4), while starter and finisher diet (B1) for G5 (Table 1).

Preparation of lymphokines

Salmonella typhymurium was isolated from Salmonella typhymurium-infected birds. Salmonella was grown on nutrient broth and peptone water. The dishes were incubated at 37°C for 24 hours until sedimentation occurred with small white deposits. The bacteria were then planted on selected medium such as MacConkey agar and SS agar. After the growth of bacteria on the growth media and the appearance of black spots, the bacteria were identified biochemically using API20. After determining the type of bacteria Salmonella typhymurium, the bacterial infected dose was determined by injecting of chicks by 1 × 10⁸ cfu. Based on spectrophotometric, the required concentration of the bacteria was determined at a standard level of 625 nm in length. Two groups of chicks were included, each group included 20-day-old chicks. The first group had three oral doses (ages 7, 14, 21). Other group did not give anything and considered as control group. At 35-day, T cell cells collected from spleen of infected birds, after crushing of infected spleens, T- cells were harvested by centrifugation at 1500 rpm. T-cells were then cultivated on RBM dishes with the addition of Co- A for stimulation of lymphokines secretion. Lymphokines were collected by centrifugation of filtrate at 3000 rpm according to (13).

Table 1: Residual of Aflatoxin, Ochratoxin and T2 contamination of feed collected feed mills estimated by ELISA test

ppb: part per billion.           

Preparation of viral inoculum

Newcastle disease isolate was taken from the Department of Pathology and Poultry Diseases, college of Veterinary Medicine, University of Baghdad. ELD₅₀ was identified as 10⁵ according to (13), and the sample was kept at deep freezing (-80).

Experimental design

The experiment was carried out using 250 chickens of one-day broiler, divided into five groups each group 50 chicks, treated as follow: the first group was injected 0.5 ml intraperitoneal with Lymphokines at one day, after 30 minutes vaccinated with Newcastle (ND) vaccine (La Sota strain) through drinking water, the injection and the vaccine was repeated at 10 days. The second group was injected 0.5 ml intraperitoneal with Lymphokines at one day, after 30 minutes was vaccinated only with killed ND vaccine at 1 day subcutaneously. The third group injected 0.5 ml intraperitoneal with Lymphokines, after 30 minutes the third group vaccinated Newcastle disease with the same strain kill and live vaccine through subcutaneous and drinking water respectively and was vaccinated only with the live vaccine at 10 days preceded by lymphokines. The fourth group was vaccinated with ND vaccine by drinking water at 1 and 10 days with not treated with Lymphokines and considered as positive control group. The fifth group was not vaccinated and not treated with Lymphokines considered as negative control group. All groups were challenge with local isolate of NDV (100ELD₅₀ 10⁵) at 25 days, where is the group which consume mycotoxins only.

Samples

Blood samples from the jugular vein were collected at different period 1, 7, 14, 21, 28, 35 and 42 to determine the antibody titer against Newcastle disease using ELISA and haemaaglutantination inhibition test after separation of the serum.

Oxidative status and antioxidant defense in liver and spleen

A total of five tissue samples of liver and spleen were collected from each group at 42 days after 12 hours of fasting. One gram was taken from each sample (liver or spleen), Placed in ice-cold and mixed with saline buffer (1: 9 wt/v) then crushed with ultra-turrax (T8, IKA-Labortechnik, Staufen, Germany). The concentration of the Mixture becomes 0.1 g/mL for analysis. The mixture was separated by a centrifuge (1000 rpm) for 10 minutes at a temperature of 4°C. It is then kept at -70°C until it is used to determine concentrations of (H2O2, MDA, LPO and GSH-PX) using the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, china).

ELISA examination

For determination the level of titers against ND virus vaccination in birds' protocol was performed according to ProFLOK NDV direct ELISA kit (Synbiotics-USA).

Examination of hemagglutination inhabitation

Hemagglutination inhabitation is one of the most specific tests for detection of antibodies against Newcastle disease in serum of infected or vaccinated birds the procedure has applied according to (14).

Challenge test

At 25-day, challenge test was performed using local isolate of Newcastle disease at (100 ELD₅₀ 10⁵) according to method of (13). The chicks were monitored for 10 days, and the morbidity and mortality were recorded for each group.

Statistical analysis

The final results were statistically analyzed using statistical analysis system (SAS). The means were distinguished according to the least significant differences and at a significant level (P <0.05).

Results

Immunity against Newcastle

Ten chicks were slaughtered before dividing chicks in groups in order to determine ND maternal immunity was accomplished by using ELISA and HI techniques. Titer WERE 7340.9±202.7 and 204.8±22.1 respectively. The study identified the role of lymphokines in improving the immune response to ND under effect of mycotoxins by giving 30 minute before vaccination. Table 2 shows significant differences at level (P<0.05) in Ab titre against ND in all periods. A significant increase at level (P <0.05) in Ab in the G3 followed by the (G1 and G2) groups respectively in (14, 21, 28, 35) days compared with G4 which recorded a significant decrease in Ab against ND, while G5 which is resemble the maternal immunity, it was recorded a decrease reached to zero in 14 days and continuous to 35 days. After challenge with local isolate of NDV all groups were recorded significant increase in Ab at 42 days, but G5 was recorded highly increase in Ab reach to 15792.2 ± 903.9.

Table 3 showed HI results of Abs against ND were identical to ELISA results. There were significant differences at level (P <0.05) in Ab titre against ND for all periods. There was A significant increase at level (P <0.05) in Ab in the G3 followed by the (G1 and G2) groups respectively in (14, 21, 28, 35) days compared with G4 which recorded a significant decrease in Ab against ND, while G5 which is resemble the maternal immunity, it was recorded a decrease reached to zero in 14 days and continuous to 35 days. After challenge with local isolate of NDV all groups were recorded significant increase in Ab at 42 days, but G5 was recorded highly increase in Ab reach to 415.4 ± 15.5.

The oxidative status and antioxidant defense

Table 4 showed the oxidative status and antioxidant defense in chicks vaccinated against ND and treated with SILK under effect of mycotoxin reassembly by (H2O2, MDA, LPO and GSH-PX) in liver tissues, at 42 days after challenge with local isolate NDV. The third group showed a significantly decrease (P>0.05) in levels of oxidative enzymes followed by the (G1 and G2) groups respectively, compared to (G4 and G5) groups, which recorded a significant increase (P< 0.05) in levels of oxidative enzymes, however a significant increase (P< 0.05) in levels of antioxidant enzymes (GSH-PX) in G3 followed by (G1 and G2) respectively, compared to (G4 and G5) groups were recorded a significant decrease at level (P >0.05).

Table 5 showed the oxidative status and antioxidant defense of the spleen tissue. The third group showed a significantly decrease (P>0.05) in levels of oxidative enzymes followed by the (G1 and G2) groups respectively, compared to (G4 and G5) groups, which recorded a significant increase (P< 0.05) in levels of oxidative enzymes, however a significant increase (P< 0.05) in levels of antioxidant enzymes (GSH-PX) in G3 followed by (G1 and G2) respectively, compared to (G4 and G5) groups were recorded a significant decrease at level (P >0.05).

Table 2: Effect of SILK on antibody titer against Newcastle disease in different periods by ELISA test

Number of samples: 10; Different letters mean significant difference at level of (P<0.05); LSD: less significant differences.

Table 3: Effect of SILK on antibody titer against Newcastle disease in different periods by HI test

Number of samples: 5; Different letters mean significant difference at level of (P<0.05); LSD: less significant differences.

Table 6 showed the clinical signs and mortality in groups vaccinated against ND and treated with SILK, after challenge with virulent local isolate of NDV (100ELD₅₀ 10⁵) at 25 days, there was a significant decrease (P> 0.05) in rates of morbidity and mortality in treated groups with SILK (G3, G1 and G2) groups, while not treated groups (G5 and G4) showed a highly significant increase (P< 0.05) in morbidity and mortality rates.

Table 4: Effect of SILK on oxidative status and antioxidant defense in liver exposed to mycotoxins

Number of samples: 5. Different letters mean significant difference at level of (P<0.05); GSH-Px: glutathione peroxidase; MDA: malondialdehyde; H2O2: hydrogen peroxide; LPO: lipid peroxidation. LSD: less significant differences.

Table 5 Effect of SILK on oxidative status and antioxidant defense in spleen exposed to mycotoxins

Number of samples: 5. Different letters mean significant difference at level of (P<0.05); GSH-Px: glutathione peroxidase; MDA: malondialdehyde; H2O2: hydrogen peroxide; LPO: lipid peroxidation. LSD: less significant differences.