Immunomodulating Effectsof Black Seed andOxytetracycline in Pigeons

A. S. Al-Ankari

Department of Clinical Studies, College of Veterinary Medicine, King FaisalUniversity, Al-Ahsa, Saudi Arabia

Chronic administration of oxytetracycline (OXT) (incorporated at a level of 0.05 g perkg of feed for 50 days) to pigeons, significantly decreased total leukocyte andlymphocyte counts, increased heterophil:lymphocyte ratio and lysosomal enzymeactivity, and decreased reticuloendothelial system function compared with controls.Coadministration of black seed (BS) at a level of 2.5% with OXT completely blocked theeffects elicited by OXT and produced immunostimulant effects in pigeons. The additionof BS to feed of pigeons could act as an immunoprotective agent when chronicadministration of antibiotics are considered.

Keywords Immunity, Black seed, Nigella sativa, Oxytetracycline, Pigeon.

INTRODUCTION

Doves (Columba livia), originally known as rock pigeons, are now domesti-

cated and kept as fantasy birds.[1] Antibiotics are considered one of the

important management prophylactic tools and therefore chronically adminis-

tered for prolonged periods of time in this species. This is not without danger,

since antibiotics such as oxytetracycline (OXT), ampicillin, and sulphona-

mides can induce immunosuppressive effects.[2]

Black seed (Nigella sativa) is a herbaceous plant that is a member of the

Ranuculocea family[3] that has antibacterial properties.[4] An immunostimu-

lating effect of the plant such as induction of T-cell leukemia polypeptides and

stimulation of interleukin-III and interleukin 1-b that affect phagocytic cells[5]

also has been demonstrated.

Immunopharmacology and Immunotoxicology, 27:515–520, 2005

Copyright D 2005 Taylor & Francis Inc.

ISSN: 0892-3973 print / 1532-2513 online

DOI: 10.1080/08923970500242327

Address correspondence to A. S. Al-Ankari, Department of Clinical Studies, College ofVeterinary Medicine, King Faisal University, P.O. Box 55055, Al-Ahsa 31982, SaudiArabia; E-mail: aankari@kfu.edu.sa

Order reprints of this article at www.copyright.rightslink.com

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This study was undertaken to investigate the effect of chronic feeding of

black seed (BS) coadministered with OXT on the immunoresponse of pigeons.

MATERIALS AND METHODS

Birds and TreatmentsFully 83 20-week-old pigeons (Columba livia) were obtained from a local

breeder and used in the study. The experiment was conducted in the Avian

Unit, College of Veterinary Medicine, King Faisal University, Al-Ahsa, Saudi

Arabia. The birds were kept in cages in temperature-controlled rooms at 23�C,

24 hr per day. Birds were fed either basal diet (control group; n = 20), diet

supplemented with 0.05 g/kg oxytetracycline (Sigma, UK, OXT group; n = 22),

2.5% crushed black seed (BS group; n = 20) or both (OXT + BS group; n = 21)

for 50 days. The basal diet was formulated using NRC[6] guidelines and

contained 19.6 g/100 g protein and 12.9 MJ/kg metabolic energy. Food and

water were given ad libitum.

Birds were bled at day 50 of the experiment. Blood samples (1 ml) were

collected from the wing vein. Serum was used for estimating lysosomal

enzymes concentrations. Heparinized blood was used for estimating reticulo-

endothelial system (RES) function.

Laboratory AnalysisTotal leukocyte counts were made using cresyl blue dye.[7] Differential

blood counts were prepared using Hema 3 stain (Fisher Scientific, Hanover

Parke, IL, USA). Activities of lysosomal enzymes such as acid phosphatase,

Table 1: Plasma concentration of oxytetracycline (OXT), lysosomal enzymeactivity (mmol/min/l) and percentage of heterophils, lymphocytes andheterophils/lymphocytes (H/L) ratio in pigeons treated with OXT and black seed(BS) for 50 days.

ParametersControl(n = 20)

OXT(n = 22)

BS(n = 20)

OXT + BS(n = 21)

Total leukocytes(1000 cells ml�1)

21.0 ± 1.3 16.1 ± 0.7* 26.1 ± 1.2* 23.6 ± 1.4

Lymphocytes % 60.3 ± 2.1 42.4 ± 2.1* 71.1 ± 3.1* 64.8 ± 3.2Heterophils % 23.7 ± 1.1 26.0 ± 1.2 25.3 ± 1.3 24.2 ± 1.1H/L % 0.39 ± 0.022 0.61 ± 0.05* 0.3 ± 0.031* 0.37 ± 0.022Acid phosphatase 7.60 ± 0.62 4.20 ± 0.53* 7.10 ± 0.61 7.20 ± 0.59Aryl sulphatase 0.93 ± 0.05 0.61 ± 0.031* 0.89 ± 0.051 0.92 ± 0.051b-glucuronidase 0.25 ± 0.021 0.13 ± 0.011* 0.26 ± 0.022 0.26 ± 0.021OXT (mg/ml) < 0.05 1.53 ± 0.15 < 0.05 1.58 ± 0.15

*p < 0.05, significantly different from controls.

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aryl sulphatase, and b-glucuronidase were measured spectrophotometrical-

ly.[8] The RES function was determined in birds by measuring the

intravascular clearance of carbon colloid as described elsewhere.[2,9] OXT

concentration in plasma was assayed using the large plate diffusion

method.[2,10] Lower detectable limit of the assay was 0.05 mg/ml. Estimates

of intra- and interassay coefficient of variation were < 10% (n = 15).

Statistical AnalysisData were expressed as means ± SD. Analysis of variance for repeated

measures using general linear model procedure of the statistical analysis

system[11] was used to test the effect of BS and OXT. Comparison of means in

different groups was made by Duncan’s multiple-range test. p < 0.05 was

accepted as statistically significant.

RESULTS

The plasma concentration of OXT increased immediately (Table 1) after the

administration of OXT to pigeons in feed. Administration of OXT significantly

Figure 1: Semilogarithmic plot of plasma carbon concentration versus time in pigeonstreated with oxytetracycline and black seed.

517Immunomodulating Effects of BS and OXT

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( p < 0.001) reduced the total number of leucocytes, and lymphocytes and

increased the heterophils:lymphocytes ratio (H/L ratio) on day 50 of the

experiment compared with controls. Activities of lysosomal enzymes were

decreased ( p < 0.05) as a result of the administration of OXT. Administration

of BS alone in feed increased leukocyte and lymphocyte counts and decreased

H/L ratio but not enzyme activities (Table 1) BS given concomitantly with

OXT blocked the changes elicited by OXT on leucocyte, lymphocyte, H/L ratio,

and enzymes activities. The values of these parameters in the BS + OXT

group were comparable to controls.

The RES phagocytic function was significantly ( p < 0.01) reduced by

OXT, but rather increased ( p < 0.01) by BS (Fig. 1). In case of simul-

taneous administration of BS and OXT, the RES function was similar to

control The carbon clearance rate or its vascular half-lives were 8.8 ± 1.2,

6.0 ± 0.6, 15.0 ± 1.7 and 9.0 ± 1.1 min for control, BS, OXT, and OXT + BS

groups, respectively.

DISCUSSION

Administration of OXT to pigeons in feed for 50 days increased the plasma

concentration of OXT and decreased the number of leukocytes, lymphocytes,

and H/L ratio. Similar immunosuppressive effects of OXT were observed in

birds, fish, and animals.[2,12–14] Further, the antibiotic depressed serum

lysosomal activity and RES phagocytic function in pigeons in a similar fashion

to that in broiler chickens.[2]

Measures of immunity that have been commonly used and assessed in

avian species included H/L ratio.[15–17] RES, and lysosomal functions.[2,8,9,18]

These parameters have been induced by BS, suggesting an immunostimulant

effect of this substance. Additionally, immunomodulating effects of BS have

been reported elsewhere.[5,19,20]

It is possible that the lack of increase in lysosomal enzyme activity during

BS administrations reflects the operation of a protective mechanism that

increases the rate at which these potentially harmful enzymes[8] are removed

from the circulation by RES and/or inhibits their normal leakage from

lysozomes. Indeed, the decrease of intravascular carbon clearance and ac-

celeration of vascular half-life, attributed to RES function, could represent one

mechanism whereby BS coadministered with OXT blocked OXT immunosup-

pressive effects.

We could conclude that BS is an immunostimulant substance via its

enhancement of lymphocytes and RES function in pigeons. Furthermore, we

suggest that incorporation of BS in feed would produce immunoprotective

effects against chronic dosing of antibiotics.

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ACKNOWLEDGMENTS

The authors are grateful to Deanship of Scientific Research, King Faisal

University, Al-Ahsa, Saudi Arabia for their support in conducting this

research and to Professor A. M. Homeida for useful suggestions during the

course of the study.

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