ANTIBACTERIAL EFFECT OF ORIGANUMVULGARE AND ASSOCIATED HEMATOLOGICAL AND SERUM BIOCHEMICAL CHANGES...

ANTIBACTERIAL EFFECT OF ORIGANUMVULGAREAND ASSOCIATED HEMATOLOGICAL AND SERUM

BIOCHEMICAL CHANGES IN CHICKENS

Kawther H. Sabah; 1 Fatma M. Yousseff 1andAshraf A. Elghoneimy2

1: Animal Health Institute

2: Department of Pharmacology, Faculty ofVeterinary Medicine,

Qena, South Valley University

ABSTRACT

The current study of discovering new antimicrobial compounds in all fields of microbial control has stimulated research regarding the Antimicrobial properties of plant compounds. The watery oregano Extract was investigated for its antimicrobial activity both in vitro and in vivo. The in vitro study was carried out to estimate the antibacterial activity of watery oregano extract against E. coli, Pseudomonas vulgarius, Enterobacter aerogenes, Salmonella species and Staphylococcus aureus isolated from 60 fecal samples of diseased and (liver, intestine, spleen and kidney) of 25 freshly dead broilers collected during an outbreak in small private poultry farm in Ismailia. The obtained results indicated that watery oregano extract was effective against all isolated testedorganisms. The in vivo study was established to

Kafrelsheikh Vet. Med. J. Vol. 7 No.1 (2009)

investigate its efficacy against experimentally inducedcolibacillosis in broiler chicks. This study was carried on 120 apparently healthy in sexed one day old broiler (Lohman) chicks. At 15th day, they were randomly allocated into main equal 6 groups (of 20 chicks each). The 1st group was non infected non treated, the 2nd was non infected oregano extract treated, the 3rd was E. coliThe obtained findings displayed that watery oregano extract was able to reduce the mortalities from 25% in infected non medicated group to 15% in infected groups treated with oregano and zero% in groups treated with ciprofloxacin and both oregano and ciprofloxacin, respectively. All infected treated groups showed a greaterreduction in clinical symptoms and postmortem lesions severity, especially in groups treated with both watery oregano extract and ciprofloxacin in combination and ciprofloxacin alone. The haematological findings revealed an increase in lymphocyte count in non infected oregano treated, infected oregano treated and infected oregano and ciprofloxacin treated groups.The biochemical study displayed that treatment of infected groups watery oregano extract, ciprofloxacin or both in combination induced an improvement in both liver and kidney functions as compared to infected unmedicated group. In conclusion our results confirm the antimicrobial potential watery oregano extract both in vitro against all isolated tested pathogenic microorganisms in this study and in vivo against experimentally induced colibacillosis in broilers. Additionally, our findings support the use of watery oregano extract in combination with known antibacterial drugs to enhance the immunity and reduce incidence of bacterial resistance. However, further research studies

Kafrelsheikh Vet. Med. J. Vol. 7 No.1 (2009) 577

are still needed to verify its antimicrobial effectiveness and to evaluate its protection against pathogens as well as to investigate its mechanism of action. Moreover, the bioassay-guided fractionation procedure to characterize and isolate the antimicrobial constituents is needed


INTRODUCTION Antimicrobial agents are mostly

synthetic chemicals and some agents are

limited to use science they cause adverse

effects on public health and reluctance by

consumers. Therefore, growing attention has

been given to natural antimicrobials which are

more readily are accepted by consumers

(Cowan,1999).Natural plant products have been

used since ancient times and their use is

increasing.nowadays. Plants are used for

medical treatment since the prehistoric

time (Dragland et al., 2003). A great number

of plant species contains various

chemical substances exhibiting health

benefit properties, anti-oxidative, anti-

inflammatory and mainly antimicrobial

effects,and their preventive and therapeutic

use with animals is increasing.

Nowadays, nearly half of the medicines

produced in the USA are originated from plants

(Cowan, 1999). Besides 40%of the Kafrelsheikh Vet. Med. J. Vol. 7 No.1 (2009) 579

prescriptions given by the doctors are

consists of plant origin medicines in

developed countries (Dragland et al.,

2003). Oregano (Origanum vulgare L.) is an

important Mediterranean herb rich in

Phenolic compounds with antioxidant and

antimicrobial activity (Matsuura et al,. 2003 and

Chun et al., 2004). It is a low-growing

perennial native to the Mediterranean,

Western Asia and North Africa. As a

culinary and medicinal herb it was already

well known in ancient Greece and Rome.

The main component of oregano oil,

carvacrol, has strong bacteriostatic and

bactericidal properties which predestine

oregano for preventive and therapeutically use

in animals (Mauch and Bilkei, 2004). As a

consequence, plant origin new materials are

extensively examined to be used instead of

synthetic feed additives (Wang and Bourne,

1998) those were in use since 1950’s. Oregano

has been used for its antibacterial (Ariana et


al., 2002; Dursun et al., 2003; Harpaz et al.,

2003 and Uslu et al., 2003),anti-inflammatory

(Youdim and Deans,2000; and Choi et al., 2002),

antioxidant (Burt and Reinders, 2003;

Botsoglu et al., 2002 and Botsoglu et al.,

2003) , insecticide (Choi et al., 2002),

antispasmodic, expectorant, fungicide

(Akgul and Ayar, 1993), antivirutic

(Cowan, 1999) properties for centuries. In

order to make use of the naturally existing

chemicals in the structure of a plant, it is

advisable to use such plants in natural forms

rather than in processed forms.

The aim of these studies were planned to

determine the in vitro antibacterial effects of natural

Origanum vulgare L crude extract (watery extract)

against bacterial pathogens isolated from

diseased and freshly dead chickens during an

outbreak in a small poultry farm in Ismailia as well

as the in vivo efficacy against induced Escherichia coli

infection. Moreover, some haematological and serum

biochemical parameters were also investigated.


Material and Methods1-First study ( Invito study):

History and clinical examination: History of

diarrhea and depression has been recorded on 250

birds out of 2000 chicks (21 day old) reared in

small private poultry farm at Ismailia

Governorate. Beside 100 birds were found dead.

Sampling:

Sixty Fecal samples from diseased

birds and specimens(liver, intestine,

spleen and kidney)of 25 freshly dead were

collected for bacteriological examinations.

Bacteriological examination: Fecal and tissue samples were

subjected to bacteriolological examination

according to Quinn, et al. (1994) and (2002). Where the

colonial morphology, staining and biochemical

reactions were done, as well as

serodiagnosis for salmonella by using

polyvalent (O) antisera Kit (Dade behring

Marburg GmbH –USA) D-35001 and for E.coli by

using (coli test serum Anti-OK(B) polyvalent


1. Antibacterial studies: Antimicrobial sensitivity test

of the isolated bacteria was done on Mueller–

Hinton medium (Oxoid) using:

A- Different chemotherapeutic sensitivity

discs (Oxoid) namely Cefotaxim (CTX - 30 µg),

Ciprofloxacin (Cip-5μg), Enrofloxacin (Enr-

10μg), Gentamycin (Cn-10μg), Doxycycline (Do-

100 μg), Cholormphenicol (C – 10 μg),

Streptomycin (S - 10 μg) , Erythromycin (E - 15

µg), and Trimethoprim/ Sulphamethoxazol (SXT-

30 μg).

B- Sterile filter paper discs of Origanum

vulgare extract: Sterile filter paper discs of

6 mm were impregnated with 20 µl of watery

Origanum vulgar extract (oregano tea 1/10 v/v).

The paper discs were allowed to evaporate and

after that placed on Mueller-Hinton plates.

Then, plates were kept for 2 hour in

refrigerator to enable diffusion of the extract

into medium.


All plates were incubated over night at 37°C.

The results were interpreted according to NCCLS

(2002) standards.

2-Second study ( In vivo study):

A-Preparation of watery Origanum vulgare extract

(oregano tea 1/10 v/v):

A 100 grams of crumbled oregano

spice (Origanum vulgar L) from a local retailer

in Ismailia, Egypt were added to 1 liter of

boiled distilled water in a sterile flask, then

the flask left for 30 minutes at 20 °C for

making oregano tea 1/10 (w/v). After this time

period, the content of the flask was heated at

95 °C for 1 minute, and filtered through

sterile cheesecloth to another sterile flask

and then, filtered through a filter paper into

another sterile flask. The content of the flask

was cooled to a room temperature and used in

the experiment

B-Experimental chicks:

One hundred and twenty apparently

healthy unsexed one day-old broiler chicks


(Lohman strain) of nearly the same weight were

used in this study. Chicks were kept in wire

floored battery brooders. They were obtained

from Ismailia Miser for Poultry Production

Company, Ismailia, Egypt. They were fed non

balanced commercial ration free from any

medicaments. They are watered adlibitum with a 24

hour light through the experimental period

C-Bacteria:

The E. coli s train O78:k80

isolated from chickens with coli septicemia

during the previous outbreak was used for

induction of experimental collibacillosis. The

bacteria were grown in a brain heart infusion

broth for 24 hours at 37 oC and then on

chocolate agar for 24 hours at 37°C. To prepare

the inoculums, the colonies were suspended in

15 ml of sterile saline at pH 7. The

concentration of the suspension was

determined by McFarland tubes. The number of

bacteria per milliliter was determined by

plating 10 fold dilutions of suspension on


plate count agar. Titers were expressed as

colony forming units per milliliter (c f u ml-1)

(Fernandez et al., 1998)

D-Experimental design:

At 2nd week of age, the chicks were

randomly allocated in 6 main groups (20 chicks

of each). Birds in groups (3, 4, 5 and 6) were

experimentally infected by injection of (1.5

X108) C F U/ ml of E. coli (O 78) suspended in

saline into the left ventral air sac. The

treatment started 48 hour post infection (after

appearance of clinical sings) and lasted for 5

successive days. The groups were named as

following:

Group (1): The birds of this group were kept

without any medications as a control group (G:

1).

Group (2): The chicks of this group were

treated with watery Origanum vulgar extract at a

dose level of (50ml/litre) for 5 successive

days (G: 2).


Group (3): The birds of this group were

infected with E.coli (1.5 X10 8) and kept

with out any treatments (G: 3).

Group (4): The chickens of this group were

infected with E.coli (1.5 X10 8) and treated

with watery Origanum vulgar extract a dose

level of (50ml/litre) for 5 successive

days (G: 4).

Group (5): The chicks of this group were

infected with E.coli (1.5 X10 8) and

treated with ciprofloxacin (1ml/litre)

(G: 5).

Group (6): The birds of this group were

infected with E.coli (1.5 X10 8) and treated

with both watery Origanum vulgar extract

(50ml/liter) and ciprofloxacin

(1ml/liter) (G: 6).

E- Clinical examination:

The birds were examined daily for clinical

signs of disease and mortality.

F-Sampling and analysis:

1-Sampling:


At 2nd, 9th and 16th day post cessation of

treatment, 5 chickens from each group were

slaughtered for sampling and two blood

samples from each bird in each group were

collected in clean dry centrifuge tubes, one

with anticoagulant (EDTA) for haematological

study and the other without any anticoagulant

(for serum collection) for clinico-

biochemical tests. The serum was separated by

centrifugation at 3000 r.p.m. for 15 minutes

and kept frozen at -20°C until analyzed.

Also, tissue samples were obtained for

bacteriological reisolation.

2-Analysis:

a)-Hematological analysis:Total leucocytic (WBCs 10³/µl) and

differential leucocytic counts were

determined according to routine hematological

examination and standard blood smear (Jain,

2000)

b)-Serum biochemical analysis: The

collected sera were assayed for serum

biochemistry. The level of alanine aspartate Kafrelsheikh Vet. Med. J. Vol. 7 No.1 (2009) 588

aminotransferase (AST) and alanine

aminotransferase (ALT) according to Reitman

and Frankel (1957), creatinine (Young et

al.,1975) and uric acid (Caraway, 1963),

total serum protein (Henry, 1964), total

albumen (Doumas, 1971) were determined by

using Auto analyzer Hitachi 912.

C)-Statistical analysis: The data were

reported as the mean ± S.E. Statistical

significance was determined using analysis of

variance according to (Snedecor and Cochran,

1982). Means were compared by Least

Significance Difference (LSD) test at 0.5

significance level Steel and Torrie ( 1980 ) .

Results and Discussion Many hundreds of plants worldwide are used

in traditional medicine as treatments for

bacterial infections (Cooke et al., 2000;

Martin and Ernst, 2003; Okoli and Iroegbu,

2004). Scientists from divergent fields are

investigating plants a new with an eye their

antimicrobial usefulness. A sense of urgency


accompanies the search as the pace as species

extinction cantinas. Laboratories of the world

have found literally thousands of

phytochemicals which have inhibitory effect on

all types of microorganisms in vitro (Cowan,

1999). The current study aimed to investigate

the in vitro efficacy of watery Origanum vulgare

extract against some prevalent isolates in

natural broilers farm outbreak as well as to

study its in vivo efficacy against

experimentally induced colibacillosis in

broilers. The affected chicks showed

enteritis, weakness and emaciation due to

anorexia (in appetence) and profuse diarrhea.

The mortality rate was 1.75%. The most

prominent isolates from diseased and freshly

dead birds were E. coli (68%). This result was

in consistence with results of Aruji et al.,

(2004) who reported that, the incidence of E.

coli isolated from diseased birds was 21%.

Also these findings support the results of

Petermanns et al., (1989), Aquirre et al.,


(1992), Tsubokura et al., (1995) and Middleton

and Ambrose (2005) they recorded that E. coli

was the commonest bacterium isolated from

organs, intestinal tract and cloacae of birds

and water fowls.

The results presented in Table 1

declared that E.coli isolation is followed in

prevalence by Pseudomonas aeruginosa (32%),

Proteus vulgaris (29%), Enterobacter aerogenes (26%),

Salmonella spp. (20%) and Staphylococcus aureus (14%)

from all samples. These results agree exactly

with that reported by Szeness et al., (1979)

who isolated Salmonella spp., Staphylococcus aureus

and Pseudomonas aeruginosa from intestinal

contents and bile of stock ducks. Kirkpatrick

and Trexler Myren (1986) who isolated

Salmonella from birds in an incidence rate of

(1.9%). Brittingham et al., (1988) who

recorded that the prevalence rate of bacterial

isolation from coloacal swabs of wild and

migrating birds were Pseudomonas spp. (22%),

Salmonella spp. (20%) and Staphylococcus spp. (15%).


Petermann et al., (1989) who isolated

Salmonella from organs (5%) and intestinal

tract (3%) of the migrating birds. They added

that Salmonella typhimurium was the most

frequently isolated Salmonella spp. Also, they

isolated Pseudomonas spp. and Staphylococcus spp.

Hubalek et al., (1998) who isolated Pseudomonas

stutzeri (4.5%) from fecal samples of migrating

birds Pseudomonas spp. was also recovered. Also,

these findings agree with Shawkey et al.,

(2005) who isolated species of the poorly

defined genus Pseudomonas from feathers of

migrating birds. They are suggesting that

birds may have acquired many of these bacteria

from the environment. Kobayashi et al., (2007)

isolated 19 strain of salmonella typhimurium from

328 cloacal swabs of birds. The serological

serotyping of isolated E. coli (Table 2) was

found to belonged to O 78 (88.89%), O1

(22.22%), O2 (20%) and O26 (8.89%). These

results were nearly similar to those reported

by Fatma Yousseff et al., (2008) who


recorded that the serotype O78 was

the most common serotype isolated

from diseased birds followed by O26, O1,

O2, O157 and O111. The obtained results of

antimicrobial sensitivity test revealed that

the watery Origanum vulgare extract was found to

be effective against all tested isolated

microorganisms in this study. Notably, the

isolated bacteria were more susceptible to the

watery Origanum vulgare extract as compared to

the standard antibiotic discs except for

ciprofloxacin and enrofloxacin. These findings

were supported by Chun et al., (2005) who

stated that Origanum vulgare L is an important

Mediterranean herb rich in phenolic compounds

with antioxidant and antimicrobial activity.

Also, these results supported by Dulger (2005)

who reported that The Origanum vulgare extracts

demonstrated antimicrobial effect against

Escherichia coli, Staphylococcus aureus, Klebsiella

pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris,

Bacillus cereus, Mycobacterium smegmatis, Listeria


monocytogenes, Micrococcus luteus, Candida albicans,

Rhodotorula rubra and Kluyveromyces fragilis.

Regarding the results of the second part of

this study, it was cleared that the

experimentally infected non treated birds

one day post E. coli (O78 of avian origin)

infection showed some clinical symptoms of

restlessness, ruffled feathers, loss of

appetite, mouth breathing and sneezing.

At 48 hour post infection, autopsy of diseased

chicks showed several gross pathological

lesions such as airsaculitis, severe lung

congestion, pericarditis, perihepatitis with

kidney and liver congestion as well as

general congestion of internal organs

indicating septicemia.These results are close

to that recorded by Barnes and Gross (1997),

Mohammad and Foud (2004) in E. coli

experimentally infected chickens, Hedawy and

Wassel (2005) and Dalia El-Shazly (2007) in E.

coli naturally infected Quails. The E. coli

(O78) experimentally infected un-medicated


birds showed some mortalities (5/20) with

mortality rate of 25%. These obtained data are

not far from that recorded by Badr (2003) who

reported that E. coli (O78) experimentally

infected non treated (20 day-old) chicks

showed a mortality rate of 40%. Also, these

findings are nearly similar to that stated by

Dalia El-Shazly (2007) who found that E. coli

(O78) experimentally infected un medicated

quails elicited some mortalities (13/40) with

mortality rate of 32.5%. The birds

experimentally infected with E.coli and

treated with oregano tea (watery Origanum vulgare

extract) displayed slight clinical symptoms

with the mortalities being reduced to

(3/20) with mortality rate of 15%. The

postmortem lesions were reduced to greater

extent as compared to infected untreated birds.

Needless to say, these findings are

confirmed by our obtained results regarding

in vitro efficacy of watery oregano extract

against tested isolated pathogens including E.


coli (Table 3), which revealed that watery

oregano extract was effective against all

tested isolated microorganisms. Oregano

(Origanum vulgare) is rich in phenolic compounds

and the highest phenolic contents

were found in watery extract. Moreover,

some phenolic phytochemicals have been

shown to have antimicrobial and

antifungal activity Chun et al., (2005).

Given these notions, one could attribute this

antimicrobial activity of watery oregano

extract to its higher phenolic

phytochemicals. In addition, this might be

owing to the immunostimulatory effect of

oregano proved by Walter and Bilkei (2004). The

birds experimentally infected with E. coli and

treated with ciprofloxacin declared

complete disappearance of clinical

symptoms, reduction in lesion scores to greater

extent with no mortalities. These results may

be attributed to the broad spectrum activity of

the drug against many pathogens. This


suggestion confirmed by the findings reported

by Sheer (1987) and Bauditz (1990) who found

that fluoroquinolones were active

against a wide range of Gram-negative, a

number of Gram-positive and mycoplasma

at low concentrations. These findings are close

to those stated by Charleston et al., (1998)

who recorded a mortality rate of 6.7% in E.coli

infected chickens treated with enrofloxacin,

Abdel-Alim et al., (2000) who reported a

mortality rate of zero % in all E.coli infected

and ofloxacin treated chicks, Dalia Ibrahim

(2005) who recorded a mortality and score

lesion of zero % in all affected organs of E.

coli infected and marbofloxacin treated birds

and Dalia El-Shazly (2007) who reported a

complete disappearance of clinical symptoms

with low mortality rate of 15% and obvious

reduction in the lesion score. The concurrent

administration of both ciprofloxacin and watery

oregano extract to E. coli (O78) infected

chickens group divulged a complete


disappearance of the clinical symptoms with

greater reduction in score lesions with no

mortalities. Needless to say, these findings

certainly may be aught to the augmented effect

of both ciprofloxacin and watery oregano

extract.

Regard concerning the haematological

findings (Table 4), the infected non treated

birds elicited a marked increase in total

leucocytic count, heterophile, monocyte

and eosinophile at 2nd and 9th day pos

cessation of treatment. These results may be

attributed to stress condition of infection.

This suggestion is confirmed by those found by

Fraser et al., (1991) who stated that

leucocytosis, lymphocytosis and monocytosis

might be associated with infection. Both

infected and non infected birds treated with

watery oregano extract showed a significant

increase in lymphocytic count at 2nd and 9th day

post cessation of treatment. These findings may

be aught to the immunostimulant effect of


oregano. This suggestion is confirmed by Walter

and Bilkei (2004) who mentioned that the

oregano has a non specific immunostimulatory

effects on immune cells.

Regarding the results of serum biochemical

analysis (Table 5), the obtained findings

displayed that, E. coli (O78) infected

un-medicated chicks elicited a significant

increase in serum AST and ALT levels. Increased

serum AST level has been associated

with hepatocellular damage in chickens,

turkeys and ducks Campbell and Coles (1986).

Serum ALT has poor specificity

for liver disease and the clinical

relevance of an increased ALT values is

decreased. For this reason, ALT frequently is

omitted from avian clinical chemistry

panels Harr (2002). The most common

cause of elevated serum AST activity in

birds is hepatic disease, while ALT is

neither a specific nor a sensitive test for

hepatocellular disease in birds and


may be useful for the detection of hepatic

disease only in carnivorous birds Thrall et

al., (2004). The infected birds treated

with ciprofloxacin displayed a

significant decrease in serum AST and ALT

values at 9th and 16th days post cessation of

treatment as compared to the infected

untreated group. These results are not far

from those of Hassan and Gaber (2005)

who recorded a significant decrease in serum

AST level (one week post

ofloxacin treatment and 2 weeks post E. coli

infection) in chickens. These findings are also

go head in head with the results of Dalia El-

Shazly (2007) who showed that

marbofloxacin treatment in E. coli infected

birds elicited a significant decrease in serum

AST level at 5 weeks old (2 weeks post

marbofloxacin treatment and 3 weeks post

infection). The E. coli infected chicks treated

with watery oregano extract only or treated

with both watery oregano extract and


ciprofloxacin denoted a significant decrease in

serum AST and ALT values as compared with

infected untreated group. Meanwhile, these

results are non significant when compared with

non infected non treated and non infected

oregano treated groups. These findings may be

attributed to the antioxidant effect as stated

by Zheng and Wang (2001), Pizzale et al.,

(2002) and Chun et al., (2005) who reported

that oregano (Origanum vulgare L) is an important

Mediterranean herb rich in phenolic compounds

with antimicrobial and antioxidant activity.

The antioxidant activity of phenolic compounds

in plants is mainly due to their redox

properties and chemical structure which can

play an important role in neutralizing free

radicals, chelating transitional metals and

quenching singlet and triplet oxygen by

delocalization or decomposing peroxides. The E.

coli infected non treated birds displayed a

significant increase in serum uric acid and

creatinine levels allover the experiment. These


findings are close to those of Mellata et al.,

(2003) who stated that avian pathogenic E. coli

belongs to extra intestinal pathogenic group of

E. coli which causes septicemia in poultry.

These results are confirmed by Dalia El-Shazly

(2007) who recorded a significant increase in

creatinine and uric acid levels in E. coli

infected non treated birds along the whole

experimental period. The infected chicks

treated with ciprofloxacin showed a significant

decrease in serum uric acid and creatinine

levels at 9th and 16th day post cessation of

treatment as compared with infected non

medicated group. These results indicating renal

tissue improvement and may be owing to the

therapeutic effect of ciprofloxacin removing

the bacteria and minimizing its damaging effect

on renal tissues. This suggestion is confirmed

by Dalia Ibrahim (2005) who reported

significant decrease in uric acid level at 4

weeks old (one week post marbofloxacin

treatment) in E. coli experimentally infected


chickens and Dalia El-Shazly (2007) who

recorded a significant decrease in serum uric

acid level at 5 and 6 weeks old (2 and 3 weeks

post marbofloxacin treatment) as compared with

infected non treated group. The E. coli

infected birds treated with either watery

oregano extract alone or both watery oregano

extract and ciprofloxacin in combination

induced significant decrease in serum uric acid

and creatinine levels at 9th and 16th post

cessation of treatment as compared with

infected non treated group, meanwhile these

changes were non significant as compared with

both non infected non treated and non infected

watery oregano extract treated groups. These

improvements may be aught to both therapeutic

effect of both ciprofloxacin as discussed

before and the kidney protective effect of

watery oregano extract owing to its antioxidant

effect of its phenolic compounds content Zheng

and Wang (2001), Pizzale et al., (2002) and

Chun et al., (2005). The E. coli infected non


treated chickens provoked a significant

decrease in serum total protein, albumin levels

(Table 6), as compared with infected non

treated group. These results indicated hepatic

damage because liver is responsible for the

production of a great proportion of plasma

protein Coles (1986). E. coli infected and

ciprofloxacin treated birds showed a non

significant change in serum total protein,

albumin and albumin at 2nd day post treatment as

compared with infected non treated group. These

findings may be attributed to liver affection

as liver is the source of total proteins Coles

(1986). The E. coli infected birds treated with

watery oregano extract or their combination

elicited non significant changes in total

proteins, albumin and globulin at 9th and 16th

post treatment as compared with non infected

non medicated group; meanwhile these changes

are significantly increased when compared with

infected non medicated group. These results

indicating improvements in hepatic functions


because of hepatoprotective effect of watery

oregano extract owing to the antioxidant effect

of its higher contents of phytophenolic

compounds as previously discussed. Table(1): Incidence of bacteria isolated from diseased and

freshly birds.

Total No. ofdiseased

and freshly dead n=85

Freshlydead

chickensn = 25

Total No. of diseased

n=60

Samples

Microorganisms%No.%No.%No.

8023.537.634.1130.6

16.47

6820322926

14

92 4048405656

231012101414

7516.6733.3331.67

20--

4510201912--

E.coliSalmonella spp.Ps. aeruginosaProteus vulgariusEnterobacter aerogenesStaphylococcus aureus

8983106Total

n= number of examined birds No.=positivenumber

%was calculated according to number (n) of examined bird.

Table (2): Serotyping of E.coli

Total no. of isolates n=45


Serogroups

No. %

O78 33 73.33O26 3 6.67O1 5 11.11

O2 4 8.89Total 45


(3): Sensitivity of the isolated bacteria to different antimicrobial agents.

Chemotherapeutic agents

E.coli Salmonella spp. Ps. aeruginosa Proteus vulgarius Enterobacter aerogenes Staphylococcus aureusNo. of strains(45) No. of strains(10) No. of strains(20) No. of strains(19) No. of strains (12) No. of strains (14)S R S R S R S R S R S R

No. %No. % No % No % No % No % No % No % No % No % No % No %

Orig.10% 43 95.6 2 4.4 9 90 1 10 19 95 1 5 18 94.7 1 5.3 12 100 -- -- 14 100 -- --

CTX 30 μg 35 77.78 10 22.2 -- -- 10 100 -- -- 20 100 17 89.5 2 10.5 8 66.6

7 4 33.33 -- -- 14 100

CIP 5 μg 45 100 -- -- 10 100 -- -- 19 95 1 5 19 100 -- -- 12 100 -- -- 14 100 -- --

Enr 10 μg 42 93.3 3 6.67 9 90 1 10 18 90 2 10 19 100 -- -- 12 100 -- -- 14 100 -- --

CN 10 μg 37 82.2 8 17.8 8 80 2 20 17 85 3 15 18 94.7 1 5.3 10 83.3 2 16.7 14 100 -- --

DO 100 μg - - 45 100 6 60 4 40 14 70 6 30 -- -- 19 100 -- -- 25 100 -- -- 14 100

C 10 μg - - 45 100 9 90 1 10 12 60 8 40 -- -- 19 100 -- -- 25 100 -- -- 14 100

S 10 μ 35 77.78 10 22.2 1 10 9 90 11 55 9 45 -- -- 19 100 -- -- 25 100 -- -- 14 100

E 15μg - - 45 100 5 50 5 50 -- -- 20 100 16 84.2 3 5.8 10 83.3 2 16.7 -- -- 14 100

SXT 30 μg 37 82.2 8 17.8 -- -- 10 100 -- -- 20 100 -- -- 19 100 -- -- 12 100 11 78.56 3 21.4

Abbreviations: Orig= Origanum extract in petroleum ether 10% , CTX: Cefotaxim, Cip: Ciprofloxacin, Enr: Enrfloxacine, CN: Gentamycine, Do: Doxycycline, C: Cholormphenicol, E. Erythromycin, S: Streptomycin, SXT: Trimethoprim, R= resistance, S = sensitive


Table (4): The hematological picture on broiler chicks experimentally infected with E.coli and infectedtreated groups

P≤ 0.05G 6

(Or.&cip+inf.)

G 5(Cip+inf.

)

G 4 (Or.+inf.)

G 3(infectio

n)

G 2(Or.)

G1(control)

Group

parameters

Time

0.53.78±0.2

a

3.75±0.1a

3.72±0.2a

4.02±0.

4a

2.09±0.0

3a

1.81±0.0

3b

WBCs 103/ µl1w

0.42.11±0.2

a2.3±0.2a

1.86±

0.1a

2.46±

0.3a

0.62±

0.01b

0.67±

0.01bH 103 /µl

0.21.06±0.3

b1.1±0.1b

1.11±0.1b

0.82±0.0

2c

1.40±

0.02a

1.05±

0.02bL 103 /µl

0.070.14±0.0

1a

0.16±0.0

1a

0.19±0.0

1a

0.2±0.0

1a

0.03±0.0

1b

0.04±0.0

1bM 103 /µl

0.30.47±0.0

2a

0.49±0.0

1a

0.56±0.0

3a

0.55±

0.03a

0.04±0.0

1b

0.05±0.0

1bE 103 /µl


0.41.85 ±

0.2b

1.96±0.2

9b

1.99±0.2

7b

2.34±0.1

4a

1.89±

0.03b

1.76±0.2

0c

WBCs 103/ µl2w

0.50.75±

0.3b

0.78 ±

0.4b

0.79 ±

0.5b

1.29±

0.3a

0.70±

0.1b

0.74±

0.2bH 103 /µl

0.31.00 ±

0.2b

1.06±

0.3b

1.08±

0.4b

0.68 ±

0.3b

1.20±0.

2a

0.92±

0.1bL 103 /µl

0.030.05±

0.02b

0.05±

0.03b

0.06±

0.02b

0.14±

0.01a

0.04±0.0

1b

0.05±

0.01bM 103 /µl

0.040.05±

0.01b

0.07±

0.02b

0.06±

0.03b

0.23±0.0

5a

0.04±0.0

1b

0.05±0.0

1bE 103 /µl

0.61.18±

0.09a

1.21±0.2

3a

1.25±0.2a

1.42±0.0

8a

1.37±0.

2a

1.18±0.1

2a

WBCs 103/ µl3w

0.30.37 ±

0.3a0.28± 0.3a

0.30±

0.4a

0.36±

0.3a

0.35±0.

3a

0. 37±

0.2aH 103 /µl

0.20.74 ±

0.2b

0.75±

0.4a

0.77±

0.3a

0.96±

0.2a

0.95±0.

2a

0.73±

0.3bL 103 /µl

0.10.05±

0.01a

0.05±

0.03a

0.06±

0.02 a

0.08±0.0

1a

0.04±0.0

1a

0.05±

0.01aM 103 /µl

0.050.03± 0.03±0.03±0.02±0.00.03±0.00.03±0.0E 103 /µl


0.01b0.03b0.02b1a1a1b

WBCs = Total leucocytic count H = Heterophile E = Eosinophile L = Lymphocyte M = Monocyte

ble (5): Some biochemical tests on broiler chicks experimentally infected with E.coli and infectedtreated groups

Creatinine mg/dlUric acid mg/dlALT u/lAST u/l Parameters

Groups 3w2w1w3w2w1w3w2w1w3w2w1w

0.48±0.03b0.44±0.05b0.48±0.06b5.8±0.8b6.7±0.5b4.8±0.9b14.9±0.3b15.16±0.3 b

12.96±0.2b

66.5±0.57b85.9±3.4b47.05±3.

3bGI(control

)0.50±0.03b0.54±0.02b0.55±0.06b4.9±0.5b6.5±0.4b5.6±1.1b13.6±0.2b16.16±0.

4b14.7±0.5b67.3±1.3b87.9±1.2b46.0±2.1bG 2(Or.)

0.72±0.1a1.12±013a1.00±0.1a9.8±1.2a10.6±0.5a9.4±1.0a21.2±0.8a19.32±0.3a

24.3±0.34a

127.2±1.9a

142.1±0.9a

123.6±4.5a

G 3(infection

)0.55±0.07b0.48±0.05b0.62±0.1

1b6.1±0.6b6.1±0.8b8.4±0.8b15.8±0.3b16.1±0.44b

20.88±0.2a69.8±2.4b91.2±1.1

3b121.6±1.

6aG 4 (Or.+inf.)

0.54±0.04b0.54±0.05b0.68±0.1b6.4±0.6b6.6±0.8b8.8±0.9b15.9±0.2b14.16±0.

3b

21.9±0.15a

71.1±2.98b

90.3±1.6b

120.9±0.8a

G5(Cip+inf.

)0.56±0.03b0.49±0.03b0.66±0.1b6.0±0.4b6.7±0.6b8.5±0.5b15.2

±0.3b15.2±0.5b

20.1±0.16a

68.1±2.0b92 ± 1.1b120±

0.7aG 6 (Or.&cip+ inf.)

0.20.230.232.51.92.72.32.25.27.45.9812.46P≤ 0.05


Table (6): Protienogram on broiler chicks experimentally infected with E.coli and infectedtreated groups

A/G ratioglobulin gm/dlAlbumin gm/dlTotal protein gm/dl Parameters

Groups 3w2w1w3w2w1w3w2w1w3w2w1w

1.07±0.0

4a

1.1±0.0

5b1.9±0.2a

1.44±0.0

4a1.35±0.1b1.1±0.09b1.54±0.1a1.48±0.1a2.1±0.04a

2.98±0.0

2a

2.83±0.0

1a

3.2

±0.03a

GI(control)

0.9±0.04a0.8±0.0

5b1.3±0.2a

1.54±0.0

4a1.65±0.1b1.5±0.09a1.4±0.1a1.35±0.1a2.0±0.04a

2.94±0.0

2a

3.00±0.0

1a3.5±0.03a

G 2(Or.)

1.4±0.02b2.2

±0 .05a1.9±0.03a0.8±0.1b0.54±0.1c

0.73±0.1

1c1.1±0.02b1.2±0.03b1.38±0.02b1.9±0.03b1.74±0.1b2.11±0.1b

G 3 (infection)

0.94±

0.1a

0.96 ±

0.11b2.1±0.04a1.56±0.1a1.46±0.1a

0.75±0.0

6c

1.46±0.0

2a1.41±0.1a

1.55±0.0

5b

3.02±0.0

1a

2.87±0.0

2a2.3±0.1b

G 4 (Or.+ inf.)

0.8±

0.04a

0.98 ±

0.06b1.8±0.07a1.58±0.1a

1.47±0.0

5a

0.8

±0.04c1.42±0.04a

1.44±0.0

5a1.4±0.05b

3.00±0.0

1a

2.91±0.0

2a2.2±0.02b

G 5(Cip+inf.)

0.8±

0.04a

0.98±

0.06b2.0±0.07a1.56±0.3a

1.42±0.0

5a

0.85±0.0

4c1.44±0.04a

1.42±0.0

4a1.45±0.04b

3.05±0.0

1a

2.91±0.0

2a2.3±0.1b

G 6(Or.&cip+inf.)

0.20.30.30.20.070.140.10.180. 30.10.20.5


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اف� م�ض� س8 رورة� اك�ت� ن ض� داا~ ات� ف�� كرون�� اومة� ال�مب� الات� م�ق� ع م�خ� مت� ي ح�� دة� ف� دن@� ة� ج�� ب� q0ب كرو دات� م�ب�ل ن� ص ق�� Bصائ� ي� ل�ها خ�� ة� ال�ت� ب� ب@� ا ب� ات� ال�ب� عص� ال�مرك�ب� دراسة� ب�� ة� ن�� غلق� ة� ال�مت� حوت8 ال�غلمب� طب� ال�ب� ش8 ن��

Bي� وش8 ال�مائ@ ات� الي�ردق� ب� لص ب�� خ� ط ل�مست� ي� ر ال�مب8 ي� ت@8 Bا حب8 ال�ب� ة ال�دراسة� ل�ب� ي¡�ت� ه�د� ر ج�� Bا . ات� كرون�� ال�مب�ة� ع�لي ي¡�ت� ال�دراسة� ال�معملب� ر ج�� Bد ا لة. وف�� ( و داج�� ا سم/ ال�حي� )م�عملب� ارح ال�ح� ات� ج�� كرون�� "ع�لي ال�مب�

ا دوم�ون�� كروت� ال�سب� ؛ م�ب� �ي� ول�وئ� كروت� ال�عصوي� ال�ق� ل ال�مب� ة� م�ب8 ات� ال�ممرض� كرون�� عص� ال�مب� ؛شب��ل/ ب� كروت� ال�سال�موب�� ارام�ب� لخ� س ف�� ت� كروت� الي�روت�� كروت� ال�مكورر�؛ م�ب� ة� و م�ب� ا/ ال�معوت�� ن�0 ر ي� كت� ؛ ال�ب�

ولة� م�ن ع�دد ي� ال�معر� ه�ت� ودي/� ال�د� ق� ة�60ال�عت� اف� ض�� الا~ ة� ن�� ض� ئ¡� ور مر م م�ن ط�ي� ي� ق� ة� م�ن ال�مست� ود� خ�� Bم�سحة� م�ا ة� م�ن ع�دد ود� خ�� Bم�غاء؛ ط�خال و ك�لي م�ا Bد؛ ا ات� ك�ب� ب� لي ع�ن� ل�كÆ م�ن 25ا~ وق� ود� ق� ة� ال�ت� ب8 ب�0 ور ج�د م�ن ال�طي�


ي�ر ن ال�صغ� سمي� �راح� ال�ت� ارع ف� ة�ة�اج�د مر� لب� س�ماع�ب� الا~ ة� ن�� ب� Ð@ب اضة� ال�كا ج . ال�خ� B@اب ب� jهرت� ال�ب Öظ� Bد ا .و ف��

ات� كرون�� ع ال�مب� مت� د ح�� ة� ض�� اع�لب� وش/8 ك�اي��ت� ل�ها ف�� ات� الي�ردق� ب� ة� ل�ب� ب� B@ب لاضة� ال�ما ن ال�خ� Bها ا حصل ع�لي� Ûال�مباء م�ا ك�ق� Bا . وع ال�دراسة� ة� م�وض�� هاة�ال�ممرض� م دراس�ي� د ت�� ق� سم ال�حي� ف�� ل ال�ح� لص داج�� خ� ا ال�مست� ه�د�

دام ع�دد خ� اس�ت� .120ن�� سة� ن� ر م�خ� ي� ا و غ� ن�0 اه�ر Öمة� ظ� وم واج�د س�لالة� ل�وه�مان س�لي� ن ع�مر ي�� سمي� كوت� ن�� " ك�ب�

غد مرور عها ب�� مت� ات� ح�� ت� ال�غلاج�� Bدا ع�راض� ( و48ن�� Bهور الا Öغد ظ� ي� ب�� Bال�غدوي ) ا س�اغة� م�نغد5اس�ي�مرت� ل�مدة� ر ب�� اس�ع و ال�سادش ع�ش8 ، ال�ب� �ي� ائ@ وم/ ال�ب8 ي ال�ي� . ف� رت� اة ال�ش8 ي� م�ب� ة� ف� ال�ب� ب� Ûام م�ب ن�� Bا

ج ع�دد ب@� م د� هاء م�دة� ال�غلاح ت�� ي� jحص5اب� لط )ل�لف� خ� Ûر م�ب ي� ات� دم غ� ب� د� ع�ن� ج�� Bم ا موغة� وت�� ور م�ن ك�ل م�ح� ط�ي�ل ة� )ل�لعر� سح� ن�� Bو ا ) ة� ب� B@ب ا مب� وك�ي� ي� qاس�ات� ال�ب ب� لط ل�لحصول ع�لي ال�مصل )ل�لق� خ� Ûدم م�ب ،) ال�دم�وي�

ل م�غدل لب� ق� وش8 ع�لي ب�� ات� الي�ردق� ب� Bي� ل�ب� لص ال�مائ@ خ� درة� ال�مست� ج ف�� B@اب ب� jهرت� ال�ب Öظ� Bد ا (./ و ف�� ي�ري� كت� ال�ب� ات� م�ن ب� لي 25ال�وق�� ة� ا~ ر م�غال�ح� ي� ة� ال�غ� موغة� ال�مغدت�� ال�مح� لص15% ن�� خ� ال�مست� ة� ن�� ة� ال�مغال�ح� موغة� ال�مغدت�� ال�مح� % ن��

ات� ب� Bي� ل�ب� لص ال�مائ@ خ� ال�مست� كل م�ن ن�� ة� ب�� موغة� ال�مغال�ح� ال�مح� وش8 وج�دة و ض�ف�ر ن�� ات� الي�ردق� ب� Bي� ل�ب� %ال�مائ@


هرت� ك�ل Öظ� Bد ا ا وف�� ن/ وج�دة. ه�د� لوك�ساس�ي� روف�� ي� òم�غا و ال�ست ن/ لوك�ساس�ي� ي�روف�� òار ال�ست وش8 و ع�ق� "الي�ردق�

ة� و ال�مغال�ج� موع�ات� ال�مغدت�� اءة�ال�مح� ب� õ¡ب Bة� ا ب� ار ال�مرص�� ن@8 öالا Æل�ك ع�راض� و ك�د� Bي� ج�دة� الا دا ف� دن@� ا س�8 اض�� ق� ح� " اب�� "

ات� ب� Bي� ل�ب� لص ال�مائ@ خ� ال�مست� كل م�ن ن�� ة� ب�� موغة� ال�مغال�ح� ال�مح� لكÆ ن�� ص ن�� خ�� Bالا ة� و ن�� حب� رب�� ش8 ة� ال�ت� ال�صق�د ا و ف�� ن/ وج�دة. ه�د� لوك�ساس�ي� روف�� ي� òار ال�ست و ع�ق� Bم�غا ا ن/ لوك�ساس�ي� ي�روف�� òار ال�ست وش8 و ع�ق� "الي�ردق�

ة� و اوت�� مق� ة� ال�لي� ا ال�دم�وت�� لان�� ي� ع�دد ال�خ� حة� ف� ة� واص�� وت�� ادة� م�عي� ن@� ة� ج�دوت8 ر� حوض�ات� ال�دم�وت�� هرت� ال�ف� Öظ� Bا

ة� موغة� ال�مغدت�� ، ال�مح� وش8 ات� الي�ردق� ب� ة� ل�ب� ب� B@ب لاضة� ال�ما ال�خ� ة� ن�� ة� وال�مغال�ح� ر م�غدت�� ي� موع�ات� ال�غ� ي� ال�مح� ل�كÆ ف� د�و ات� الي�ردق� ب� ة� ل�ب� ب� B@ب لاضة� ال�ما ال�خ� ة� ن�� ة�ش8وال�مغال�ح� ب� B@ب لاضة� ال�ما ال�خ� ة� ن�� ة� وال�مغال�ح� موغة� ال�مغدت�� وج�ده�ا وال�مح�

هرت� Öظ� Bد ا ق� ة� ف�� ب� B@ب ا مب� وك�ي� ي� qحوض�ات� ال�ب م�ا ال�ف� Bا . ن/ م�غا لوك�ساس�ي� ي�روف�� òار ال�ست وش8 وع�ق� ات� الي�ردق� ب� "ل�ب�

ن/ ك�لا لوك�ساس�ي� روف�� ي� òار ال�ست وش/8 وع�ق� ات� الي�ردق� ب� ة� ل�ب� ب� B@ب لاضة� ال�ما دام ال�خ� خ� اس�ت� ن ال�غلاح ن�� Bها ا ج� Bاب� ب� jب�"

د وال�كلي ف� ك�لا م�ن ال�كب� Bائ� Öي� وظ� حسن م�لحوظÖ ف� ج�دات8 ب�� درة� ع�لي ا~ و ك�لاه�ما م�غا لة ال�ق� Bع�لي ج�دة ا" "

لاضة� درة� ال�خ� د ف�� ك�ب� Bا لي ن�� ة ال�دراسة� ا~ لصب� ه�د� د ج�� ا وف�� .ه�د� ة� ر م�غال�ح� ي� ة� وال�غ� موغة� ال�مغدت�� ال�مح� ة� ن�� ارت�� م�ق�


ة ع�لي ض�د درت�� ل�كÆ ف�� ا وك�د� ولة� م�عملب� ات� ال�معر� كرون�� ع ال�مب� مت� ط ح�� ي� qب ب8 Ûوش8 ع�لي ب� ات� الي�ردق� ب� ة� ل�ب� ب� B@ب "ال�ما

ة د دع�مب� ه�د� ا وف�� . ه�د� ن سمي� داري� ال�ت� ي� ن�� ا ف� ب� qب òج�رت� ة� ب�� ي� ال�عصوي� ال�مخدت�8 ول�وئ� كروت� ال�ق� ع�دوي ال�مب�ادا وش8 م�ع ال�مص� ات� الي�ردق� ب� ة� ل�ب� ب� B@ب لاضة� ال�ما دام ال�خ� خ� ة� اس�ت� ب� ب@� م�كا ة�ت�ال�دراسة� ا~ ة� ال�معروف� �ÿت ر ي� كت� ال�ب�

ادا لكÆ ال�مص� ة� ل�ب� �ÿت ر ي� كت� اومة� ال�ب� ة� م�ع ال�خد م�ن ج�دوت8 ال�مق� اع�لب� اغة� وال�ق� ادة� ال�مب� ن@� ل�كÆ ل�ر� ات�ود� . ه�د�ا اءة� ه�د� د ك�ق� ك�ب� Bا ة� ل�ب� لب� ب� ق� د م�ن ال�دراس�ات� ال�مست� راء ال�غدن@� ج�� لي ا~ ة� ا~ اك�Æ ال�خاج� ال�ت� ه�ب� وم�ار�

ان ب� qب ة/� واس�ب� ات� ال�ممرض� كرون�� ي� ض�د ع�دوي ال�مب� ة ف� اءت�� م ك�ق� ي� ن� ق� ل�كÆ ب�� ات� وك�د� كرون�� اد ل�لمب� لاضة� ك�مص� ال�خ�ل د وع�ر� خدن@� Ûلص ل�ب خ� ا ال�مست� ات� ه�د� صل م�كون�� ة� ف�� لي ع�ملب� ة� ا~ اك�Æ ج�اج� ن ه�ب� Bة� ال�عمل. ك�ما ا ب� ق� ك�ت�

ا. ول ع�ن ه�د� Bال�مسي ال�مكون


ANTIBACTERIAL EFFECT OF ORIGANUMVULGARE AND ASSOCIATED HEMATOLOGICAL AND SERUM BIOCHEMICAL CHANGES...

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Transcript of ANTIBACTERIAL EFFECT OF ORIGANUMVULGARE AND ASSOCIATED HEMATOLOGICAL AND SERUM BIOCHEMICAL CHANGES...