International Scholarly Research Notices

International Scholarly Research Notices / 2013 / Article

Research Article | Open Access

Volume 2013 |Article ID 849412 | https://doi.org/10.1155/2013/849412

Faten A. Okda, Safaa Yassein, Alaa R. Ahmed, Hamdy Soufy, Soad M. Nasr, "Some Haematological and Biochemical Investigations on Duck Virus Hepatitis following Administration of Glycyrrhizin", International Scholarly Research Notices, vol. 2013, Article ID 849412, 10 pages, 2013. https://doi.org/10.1155/2013/849412

Some Haematological and Biochemical Investigations on Duck Virus Hepatitis following Administration of Glycyrrhizin

Academic Editor: T. W. Stone
Received19 May 2013
Accepted02 Jul 2013
Published30 Jul 2013

Abstract

The present study aimed to investigate the protective effect of glycyrrhizin (locally isolated and purified from licorice root) against duck hepatitis virus through the assessment of some hematological and biochemical parameters. One hundred and sixty white Pekin ducklings—one day old—were randomly divided into four equal groups. Group (1) was kept as normal control. Group (2) was inoculated I/P with 10 mg glycyrrhizin/kg BW, three times per week for four weeks. Group (3) was inoculated I/M with 0.5 ml of live attenuated DHV vaccine. Group (4) was inoculated with both glycyrrhizin (10 mg/kg BW I/P, three times per week for four weeks) and live attenuated DHV vaccine (0.5 ml, I/M). Then, all groups of treatment were challenged using virulent DHV except for 20 ducklings from the normal control group which were continued to be kept as negative control. The results revealed that duck hepatitis virus (DHV) caused macrocytic hypochromic anemia, leukopenia, hypoproteinemia, hypoalbuminemia, hyperglycemia, hypercholesterolemia, and marked elevation of liver enzymes and renal parameters. In conclusion, glycyrrhizin injected alone or in combination with DHV vaccine protected or ameliorated the deteriorating effects induced by DHV vaccine and/or duck hepatitis virus infection by improvement of erythrogram and leukogram, as well as liver and kidney functions.

1. Introduction

Duck virus hepatitis (DVH) is one of the most economic import diseases to all duck-growing farms because of its high potential mortality if the infection is not controlled. It is an acute highly fatal rapidly spreading viral infection of young ducklings. It was first recoded in New York and Taiwan. The morbidity is 100% and the mortality may reach 95–100%, in the first week of age [1].

Survived ducklings after DHV infection have a solid immunity, but it is necessary to protect the duck industry against such fatal disease using the potent-specific vaccine. Live attenuated DVH-1 vaccine which could be administrated through the intramuscular route in breeder ducks 2-3 weeks before lying allowing the transmission of high maternal immunity to the offspring providing them with passive immunity that is able to protect the new hatched birds up to 15 days of age. Also, it could be injected in 2-day-old ducklings followed by a booster dose 2-3weeks later [2].

In Egypt, duck farms are routinely protected against DVH following a vaccination program employing DVH-1 which is the only vaccine recorded in Egypt. Unfortunately, some duck farms stopped vaccination against DVH causing the recurrence of disease outbreaks [1].

Glycyrrhizin (GL) is the major active component extracted from licorice (Glycyrrhiza glabra) roots. GL has anti-inflammatory and antiviral effects that have been used in the treatment of patients with chronic hepatitis B and C [37]. GL enhances the production of antibodies through the production of interleukin- 1 (IL-1), IL-2, and IL-12 [8]. Soufy et al. [9] recorded that GL possesses a good immunostimulant and synergistic effect to DVH vaccine through activation of T lymphocyte proliferation. GL with vaccine enhances higher antibody titer against DHV than vaccine alone and high immunity protection.

The present study was conducted to investigate the protective effect of glycyrrhizin injected alone or in combination with live attenuated DHV vaccine against experimental infection of ducklings with virulent duck hepatitis virus through the assessment of some haematological and biochemical parameters.

2. Materials and Methods

The experiment was done according to guidelines for animal experimentations and approved by the Institutional Animal Care and Use Committee, National Research Center, Animal Care Unite, Dokki, Giza, Egypt.

2.1. Extraction, Purification, and Identification of Glycyrrhizin from Licorice Plant

Two kg of licorice root (Glycyrrhiza glabra L.) was obtained from Haraz, Abdeen, Cairo, Egypt, for extraction of glycyrrhizin (GL). Purification of glycyrrhizin from licorice was performed according to Bentley and Trimen [10]. The extracted and purified substances were identified using thin layer chromatography (TLC) according to the method of Cui et al. [11].

Glycyrrhizin was administrated as stronger neo-minophagen C (SNMC). It consists of purified glycyrrhizin 2% + cysteine 0.2% + glycine 2% dissolved in physiological saline. It was inoculated 3 times weekly for 4 weeks [12]. Cysteine and glycine were added to avoid side effect of glycyrrhizin by increasing glutathione synthesis and prevent the sodium and water retention effect.

2.2. Ducklings Used

One hundred and sixty white Pekin domestic ducklings one day old, obtained from a private nonvaccinated parent flock without maternal immunity were used in this study. Ducklings were housed under hygienic measures in separate isolators receiving a balanced growing broiler ration, containing protein 21%, fats 3.6%, and fibers 3.4% according to NRC [13].

2.3. Experimental Design

Ducklings were kept for 4 days for acclimatization and were allocated into 4 equal groups. Group (1) was kept as normal control (without glycyrrhizin treatment or vaccination). Group (2) was inoculated intraperitoneally (I/P) with 10 mg of glycyrrhizin/kg BW as SNMC (3 times weekly for 4 weeks). Group (3) was inoculated intramuscularly (I/M) with 0.5 mL of live attenuated DHV vaccine (obtained kindly from Vet Serum and Vaccine Research Institute, Abbassia, Cairo). Group (4) was inoculated with glycyrrhizin as SNMC (10 mg/kg BW, I/P, 3 times weekly for 4 weeks) and live attenuated DHV vaccine (0.5 mL) I/M. At the end of the 3rd week, all groups were challenged except 20 ducklings from the normal control group were continued to be kept as negative control.

2.4. Hemogram and Biochemical Analysis

Blood samples were collected from each duckling by jugular vein puncture before the challenge test at the end of the 1st, 2nd, and 3rd weeks and after the challenge at the 4th week of the beginning of the experiment. Each blood sample was divided into two portions; the first one was anticoagulated by disodium salt of ethylene diamine tetra acetic acid (EDTA) for determination of hemogram [14]. The second portion was placed in a plain centrifuge tube for serum separation and determination of biochemical constituents; total proteins [15], albumin [16], activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) [17], alkaline phosphatase (ALP) [18], glucose [19], creatinine [20], uric acid [21], and cholesterol [22]. Test kits supplied by bioMérieux-France were used.

2.5. Challenge Test

The ducklings of each group at the end of the 3rd week of treatment (at the 25th day of age) were challenged I/M with 0.5 mL virulent DHV containing 107 TCID50 per duckling. The strain was kindly supplied from Vet Serum and Vaccine Research Institute, Abassia, Cairo.

2.6. Statistical Analysis

Results are expressed as the mean ± SD. Differences between control and treated groups and differences between control-infected group and other groups after challenge were tested for significance using a one-way analysis of variance followed by least significant difference (LSD). Differences were considered significant at level [23] using SPSS version 10 computer program.

3. Results

3.1. Clinical Signs

Before the challenge, ducklings of normal control and glycyrrhizin-treated groups (1 and 2) appeared healthy during the experimental period. However, vaccinated group (3) showed signs of illness in the form of depression, decreased food intake, ruffled feather, and dullness at the 2nd and 3rd days. These signs started to disappear at the 4th and 5th days after vaccination. On the other hand, ducklings of treated and vaccinated group (4) were slightly depressed, mildly anorexic at the 2nd and 3rd days after vaccination, and returned to normal at the 4th and 5th days.

After the challenge test (at the 25th day of age), control infected group of ducklings showed severe depression, ruffled feather, and off food. Some ducklings were lying on their sides or breast with leg extended backward and head drown over the back with spasmodic paddling leg movement on the 3rd, 4th, 5th, and 6th days after challenge. The morbidity rate reached 80%, and the mortality rate reached 70%. Glycyrrhizin group (2) showed slight depression and general signs of illness at the 2nd day after challenge; one duckling had spasmodic paddling leg movement but returned healthy at the 3rd day. Neither mortality nor morbidity was recorded, and ducklings appeared healthy with normal size. In addition, ducklings of vaccinated group (3) showed moderate depression and general signs of illness at the 2nd day after challenge; one duckling died at the 3rd day after challenge. The survived duckling was emaciated and maintained stunted growth. In contrast, high protection rate (100%), healthy appearance, normal growth, and size were observed in glycyrrhizin-treated and -vaccinated ducklings (group 4) as shown in Table 1.


GroupsMorbidity rate Mortality rate/days after infectionMortality rateProtection rate
1234567891011121314

Control 80%231170%30%
Glycyrrhizin 10%0%100%
Vaccinated 10%110%90%
Glycyrrhizin and vaccinated 0%0%100%

3.2. Erythrogram

Compared to normal control group (1) before challenge, red blood cell count (RBC), packed cell volume (PCV) %, and haemoglobin (Hb) concentration showed significant increase in glycyrrhizin-treated group (2) from the 1st till the 3rd week. Glycyrrhizin-treated and vaccinated group (4) showed significant increases in RBCs count and PCV % at the 2nd week. Vaccinated group (3) showed significant decreases in the RBCs count, PCV %, and Hb concentration at the 1st and 2nd weeks. At the 4th week after challenge, RBCs count, PCV %, and Hb concentration showed significant decrease in all groups compared to the normal control group (Table 2).


Groups
ParametersPeriods (weeks)Control (1)(2)(3)(4)LSD
NormalAfter challengeGlycyrrhizin DHV vaccineGlycyrrhizin + DHV vaccine

Red blood cell count (106/µL)1 0.32
2 0.30
3 0.37
4# 0.33

Packed cell volume (%)1 3.12
2 2.60
3 3.30
4# 2.62

Hemoglobin (g/dL)1 0.52
2 0.59
3 0.68
4# 0.91

Mean corpuscular volume (fL)1 12.50
2 12.47
3 10.70
4# 14.47

MCHC (g/dL)1 2.81
2 2.15
3 1.05
4# 2.51

4# After challenge (at the 4th week).
Significant compared to control normal group in the same row.
Significant compared to control-infected group at the 4th week.
DHV: duck hepatitis virus.
LSD: least significant difference.
MCHC: mean corpuscular hemoglobin concentration.

Vaccinated group (3) showed significant an increase in mean corpuscular volume (MCV) and a significant decrease in mean corpuscular haemoglobin concentration (MCHC) at the 1st week before challenge in comparison with normal control group (1). At the 4th week after challenge, significant increase in MCV with marked decrease in MCHC was demonstrated in control-infected and vaccinated groups, while no significant changes were observed in glycyrrhizin-treated group (2) and glycyrrhizin-treated and -vaccinated group (4). This data revealed the presence of macrocytic hypochromic anemia at the 1st week in vaccinated group and at the 4th week (after challenge) in control-infected and vaccinated group (Table 2).

3.3. Leukogram

Before challenge, compared to normal control group (1), glycyrrhizin-treated group (2) showed significant leukocytosis all over the experimental period with lymphocytosis started from the 1st week and monocytosis started from the 2nd week till the end of the experiment. Vaccinated group (3) showed significant leukopenia at the 1st week associated with significant lymphocytosis at the 3rd week and monocytosis from the 1st week till the end of experiment, while significant decreases in heterophil and eosinophil count were detected from the 1st week till the end of the experiment. Glycyrrhizin-treated and -vaccinated group (4) showed leukocytosis, lymphocytosis, and monocytosis from 1st week till the end of the experiment (Table 3). At the 4th week (after challenge), compared to control negative group, significant leukopenia, heteropenia, and eosinopenia were detected in control-infected, vaccinated, treated and vaccinated, and treated groups, respectively, with significant lymphocytosis and monocytosis in glycyrrhizin-treated and -vaccinated, control-infected, and treated groups, respectively (Table 3).


Groups
ParametersPeriods (weeks)Control (1)(2)(3)(4)LSD
NormalAfter challengeGlycyrrhizin DHV vaccineGlycyrrhizin + DHV vaccine

Total leukocytic count (103/µL)1 1.70
2 1.60
3 1.86
4# 3.69

Lymphocyte count ( 103/µL)1 4.74
2 5.64
3 4.01
4# 1.89

Heterophil count
( 103/µL)
1 1.32
2 0.42
3 0.78
4# 0.49

Monocyte count ( 103/µL)1 0.19
2 0.20
3 0.10
4# 0.10

Eosinophil count
( 103/µL)
1 0.10
2 0.12
3 0.07
4# 0.00

4# After challenge (at the 4th week).
Significant compared to control normal group in the same row.
Significant compared to control-infected group at the 4th week.
DHV: duck hepatitis virus.
LSD: least significant difference.

3.4. Serum Proteins Profile

Before challenge, compared to normal control group (1), glycyrrhizin-treated group (2) showed significant increases in total proteins and globulins at the 2nd week with significant increases in albumin at the 2nd and the 3rd weeks. Vaccinated group (3) showed significant increase in total proteins with significant hyperglobulinemia at the 2nd week, while significant hypoalbuminemia was observed at the 1st and 2nd weeks with significant decrease in A/G ratio from 1st week till the end of the experiment. Glycyrrhizin-treated and -vaccinated group (4) showed significant increase in total proteins with significant hyperglobulinemia at the 2nd and 3rd weeks, while significant decreases were observed in A/G ratio from the 1st week till the end of the experiment except for significant increase at the 3rd week with no significant changes in albumin concentration (Table 4). At the 4th week (after challenge), control-infected group showed significant hypoproteinemia, hypoalbuminemia, and significant decreases in A/G ratio compared to normal control group (1). Glycyrrhizin-treated group (2) showed significant increase in total proteins with hyperglobulinemia and significant decrease in A/G ratio at the 4th week and no significant changes in albumin. Vaccinated group (3) and treated and vaccinated group (4) showed significant hyperglobulinemia, hypoalbuminemia, and significant decrease in A/G ratio at the 4th week (Table 4).


Groups
ParametersPeriods (weeks)Control (1)(2)(3)(4)LSD
NormalAfter challengeGlycyrrhizin DHV vaccineGlycyrrhizin + DHV vaccine

Total proteins (g/dL)1 0.63
2 0.67
3 0.61
4# 0.58

Albumin (g/dL)1 0.23
2 0.20
3 0.27
4# 0.20

Globulins (g/dL)1 0.47
2 0.20
3 0.22
4# 0.37

A/G ratio1 0.10
2 0.20
3 0.04
4# 0.10

Total cholesterol (mg/dL)1 34.98
2 34.56
3 22.63
4# 81.00

Glucose (mg/dL)1 43.02
2