STUDIES ON THE EFFECT OF THYME OIL ON SALMONELLA BACTERIA IN BROILER CHICKS

STUDIES ON THE EFFECT OF THYME OIL ON SALMONELLA BACTERIA IN BROILER CHICKS

MONA A. AHMED^*; FATMA M. YOUSSEF^** and ABDEL RAHMAN, A.G^*

^* Animal Health Research Institute, Alarish

^** Animal Health Research Institute, Ismailia

Email: fatmayousseff@yahoo.com

INTRODUCTION

Salmonella is one of the most important pathogenic genera implicated in food borne bacterialdiseases both in developed and developing countries and constitute an important public health problem (Erdem et al., 2005). Despite global improvements in public health facilities, a marked increase in human salmonellosis has been reported in many countries including the European Union (National disease surveillance center, 2004).

Salmonellosis account for a severe problem of public health both in developing and developed countries and several controlling mechanisms of such pathogen have been applied to poultry production (Cardoso and Carvalho, 2006). More than 2500 serotypes of Salmonella have been identified (Grimont and Weill, 2007) of which Salmonella enteritidis, Salmonella typhimurium and Salmonella heidelberg are the most prevalent serotypes (Schlundt et al., 2004). Hematological and biochemical alterations recorded the severity of infection of Salmonella typhimurium. Immunological parameters revealed anincrease in serum alpha, betaglobulins anda significant decrease in Phagocytic activity and Phagocytic index (Salim     et al., 2011).

Thyme (Thymus vulgaris) is a member of Lamiaceae family, with the main components of Phenols, thymol (40%) and carvacrol (15%) (Mikaili et al., 2010). This species has special functions such as antispasmodic, expectorant, antiseptic, antimicrobial and antioxidant (Abu-Darwish and Abu-Dieyeh, 2009 and Mikaili et al., 2010). Thymol and carvacrol are the main antibacterial active substances, so this plant can be used instead of commercial antibiotics. The beneficial value of thyme in poultry industry has been reported (Demir et al., 2008). Research carried out in Sicily (Biondi et al., 1993), Sardinia (Frink    et al., 1999) and Albania (Miceli et al., 2002) showed that the thymecontains carvacrol and small quantities of thymol, often lower than 1%. There is a limited data of the beneficial effect of essential oils from thyme on microbial load as well as of specific pathogen like Salmonella on broiler carcasses (Aksit et al., 2006).

This  study  was  planned  to  isolate  and  identify  the  prevalent Salmonella species  in broiler chicken flocks  and investigate the effects of supplementation of Thyme (Thymus vulgaris) on Salmonella infection in broiler chickens and the improvement of immunity of birds and function of liver and kidney as well as hematological picture.

MATERIALS and METHODS

Poultry: A total of 100 broiler chicks (50 diseased and 50 freshly dead) were collected from broiler chicken flocks at Ismailia governorate which suffered from diarrhea with high morbidity and increased mortality. The clinical signs and postmortem lesions were recorded.

Samples: A 50 cloacal swabs and 300samples of liver, intestine, spleen, kidney, heart and gall bladder from recently dead chicks were collected for bacteriological examination.

Isolation, identification and serotyping of Salmonella: Salmonella cultures from all samples were performed according to Waltman (1999). Briefly, samples collected in buffered peptone water (BPW) were taken to the laboratory on the day of collection under refrigeration with minimum of delay and incubated at 37 °C for 24 h. After pre-enrichment, 0.1 ml of the broth culture was transferred into a 10 ml Rappaport–Vasilliadis broth (RV broth, Difco, USA) and was incubated at 42°C for 24–48 h. The RV broth samples were streaked onto Xylose-Lysine-Desoxycolate agar (XLD, oxoid) plates, brilliant green gar, SS agar, MacConkey agar, blood agar and EMB agar and incubated overnight at 37°C.The typicalcolonies were picked and further tested by standard biochemical methods (Quinn et al., 2002) andsero-diagnosis for salmonella by using polyvalent (O) and monovalent   antisera Kit (Dade Behring Marburg GmbH –USA) D-35001.

Thyme oil: It was purchased from Alarish shop. Liquid samples, which include thyme oil, were prepared as 20% (vol/vol) solutions in 80% ethanol. These were considered stock solutions and were diluted accordingly, depending on the assay used. Dilutions of at least 10 minimized the effects of ethanol so that the effects of ethanol were considered negligible. Thymus vulgaris alcoholic extract was prepared using the standard maceration method (Zhang et al., 2005) that have been reported some elsewhere (Abdulkarimi et al., 2011).

Antibacterial sensitivity test: 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), Chloramphenicol (C – 10 μg), Streptomycin (S - 10 μg), Erythromycin (E - 15 µg), and Trimethoprim/ Sulphamethoxazol (SXT-30 μg).

B- Sterile filter paper discs of Thymus vulgaris oil extract: Sterile filter paper discs of 6 mm were impregnated with 20 µl of oily thyme vulgar extract. The paper discs were allowed to evaporate and after that placed on Mueller-Hinton plates; 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.

Experimental design:

One hundred broiler chicks (Hubbard breed) one day old were used in this study. Birds were randomly divided into five equal groups. Each one contained 20 birds. All birds were subjected to the ordinary vaccination program for broilers against Newcastle and Gumboro diseases. All birds were fed balanced commercial starter, growing rations (21% and 18% protein, respectively) and fresh clean drinking water. The birds were housed in floor – pen (0.1 m2 / bird), in clean well ventilated separate experimental rooms throughout the period of experiment. Birds groups: Control Group; non-treated normal chicks(G1), (prebiotic group); normal chicks treated with thyme oil (2ml/kg feed additive)(G2), (Salmonella typhimurium infected group); infected non-treated chicks(G3), (infected treated group); infected chicks and treated with thyme oil (G4), and finally (infected treated group); infected chicks and treated with ciprofloxacin (5mg/kg body weight in drinking water for 5 successive days) (G5). Chicks of G3,G4 and G5 were infected orally with 0.2 ml of an inoculum  containing approximately (1.5×10⁸ CFU/ml)of Salmonella typhimurium directly into the crop using crop tubesat the 7^thday of age. After that trials to re-isolation of Salmonella typhimurium fromfeces and internal organs of all inoculated chicks were carried out.

Colony Count of Salmonella typhimuriumin Feces of chicks: One gram of feces was freshly collectedfrom each group separately; feces were weighed and diluted in regenerated sterile buffered saline (pH 7.2). Viable Salmonella typhimurium organisms were determined (Silva et al., 1999).

Samples Preparation for colony count: liver, spleen, cecal pouch, and lung samples of 25 g were removed aseptically and homogenized in 225 mL of 0.1% sterile peptone water for 2 min using a Stomacher 400 Lab Blender to provide dilution of 10^-1. From the original homogenate, 1 mL was transferred aseptically to a test tube containing 9 mL sterile 1% peptone water (w/v) to prepare dilution of 10^-2, then from which further tenfold decimal serial dilution up to 10^-8 were prepared. From these serial dilutions, the bacteriological investigations were performed.

Microbiological Evaluation: Appropriate diluents of each tube were placed on the following media in duplicate; Plate Count Agar (Difco Co., Ltd.) for total salmonella counts.

Blood samples: Blood was collected from wing veinof10 birds in each group at 3 ,7 and 14 days post infection and divided as follow: EDTA blood: Blood was collected in clean dry bottle containing dipotassium salt of EDTA as anticoagulant in concentration of 2mg/1ml of blood (Jain, 2000) and used for hematological studies.

Serum: blood was collected in plain clean well-dried centrifuge tube and used for separation of serum to be used in estimation of biochemical parameters.

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 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), albumen (Doumas, 1971) were determined by using  Auto analyzer Hitachi 912.

C)-Immunological study:

1. Serum protein electrophoresis: It was performed according to the method described by Markovicva    et al. (2009).

2- Newcastle Disease Vaccine Response: Newcastle disease vaccine (LaSota) wasadministrated in the drinking water at 10 days of age. Blood samples were collected on 7and 14 days after vaccination and antibody response was determined by using HImethod.

Statisticalanalysis: After obtaining the data, they were analyzed by variance method (ANOVA) considering P < 0.05 using SPSS 18.0 software. The significant differences were taken to Duncan multiple range tests to compare the means.

RESULTS

Clinical signs: young chicks suffering from acute form of disease are usually found dead. Sick chicks exhibit listlessness, dehydration and diarrhea. Other showed signs of disease, such as depression, weakness, loss of appetite, ruffled feathers, dropped wings, and closed eyes. The gross lesions in the liver showed variable changes including friable with white necrotic foci (Fig, 1&2). Lungs were congested and pneumonic (Fig, 3), spleen was enlarged (Fig.4), pericarditis (Fig.5), Intestine showed various grades of enteritis with mucous in the lumen and unabsorbed yolk sac (Fig.6).

Incidence of salmonella is presented in Table 1. Identification of salmonella is based on conventional bacteriological and biochemical characters (Table 2). A 13 salmonella isolates were identified and belonged to serotype, namely salmonella Typhimurium by using polyvalent and monovalent specific Salmonella antisera.

The results of the in vitro antibiogram test are summarized in Table (3). The obtained results of antimicrobial sensitivity test revealed that thymus vulgaris extract was found to be effective against the isolated tested microorganisms (Fig.7&8). In this study, notably, the isolated bacteria were more susceptible to the oily thymus vulgaris extract in comparison the standard antibiotic discs except for ciprofloxacin and enrofloxacin.

In the results of the second part of this study, it was clear that the experimentally infected  non treated birds three day post salmonella typhimurium infection showed some clinical symptoms of restlessness, ruffled feathers, loss of appetite, mouth breathing and sneezing. At 7^th day post infection, autopsy of diseased chicks showed several gross pathological lesions such as lung congestion and pericarditis (fig.3&5), enlarged liver with altered color (Fig. 9), hemorrhagic spots on kidneys (fig. 11) as well as pin point hemorrhage with thickened wall of intestine (fig. 12). The treated group with thyme extract showed improvement in lesion in compared to infected non treated group (Fig.10).

There was mortality reported in thyme fed group (15%) and no mortality in ciprofloxacin treated group. On the other hand, salmonella experimentally infected un-medicated birds showed some mortality (6/20) with mortality rate of 30%. The birds experimentally infected   with salmonella and treated with thyme oil 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. The birds experimentally infected with salmonella typhimurium andtreated with ciprofloxacin declared complete disappearance of clinical symptoms and reduction in lesion scores to greater extent with no mortalities. Re-isolation of the microorganism from the different inoculated chicks were down from cloacal swabs at 1, 2, 3 and 7 DPI salmonella typhimurium was consistent in being the highest in re-isolation through the 7 days of observation (100% on the 7^th days DPI) from the liver, cecal pouch, spleen and lung of infected chicks.

The results of Salmonella typhimurium colony count from fecal and internal organs samples of chicken in non-treated and treated experimental groups (table 4&5) showed the decrease in number of colony count after treatment by thyme extract and ciprofloxacin. These results indicated bactericidal effect of thyme on salmonella.

Regarding theleukogram findings (Table 6), the infected non treated birds, thyme feeding group and treated group elicited a marked increase in total leucocytic count, heterophil, monocyte and eosinophil at the 3^rdandthe 7^thday post infection. Onthe other hand on the 14^thdays post infection elicited asignificant increase in total leucocytic count, lymphocyte in infected non treated group but in the other group showed no changes in leucocytic count and differential leucocytic cells.

The results of serum biochemical analysis (Table 7), the obtained findings displayed that the infected un-medicated chicks elicited a significant increase in serum AST and ALT levels. The infected birds treated with thymus extract and the infected group and treated with ciprofloxacin displayed a significant decrease in serum AST and ALT values at the 7^th and the 14^th days post treatment as compared to the infected untreated group. Meanwhile, these results are non-significant when compared tocontrol group and non-infected thyme treated groups.

The infected non treated birds displayed a significant increase in serum uric acid and creatinine levels allover the experiment. The infectedchicks treated with thyme extract and ciprofloxacin induced significant decrease in serum uric acid and creatinine levels at the 3rd and the 14^th day post infection as compared to infected non treated group.

The infected non treated chicks provoked a significant decrease in serum total protein and albumin levels and significant increase in globulin (Table 8) as compared tocontrol group at the 3rd day post infection. The ciprofloxacin treated chicks showed a non-significant change in serum total protein and albumin at the 3^rdday post infection as compared to infected non treated group. Thyme treated group with infection were elicited non-significant changes in total proteins and albumin with increased in globulin at the 7^th and the 14^th post infectionas compared to non-infectednon-medicated group. There was significant increase in serum alpha, beta and gamma globulins of thymegroup compared to the control group. While there was significant increase in serum alpha and beta globulins of infected non treated group and infected treated groups with thyme extract compared to control group (Table 8).

There was significant increase in antibody titer in group 2 compared to the control group (G1). Also, there was significant increase in antibody titer in group 4 when compared to group 3 (Table 9).

Table 1: The prevalence of Salmonella isolated from natural diseased and dead broiler chicken.

Table 2: Biochemical characters of suspected Isolates of Salmonella typhimurium.

    Positive        (+)         Negative    (-)

Table 3: Sensitivity of the isolated Salmonella typhimurium to different antimicrobial agents.

Abbreviations:     CTX: Cefotaxim, Cip: Ciprofloxacin, Enr: Enrofloxacin, CN: Gentamycin,   Do: Doxycycline, C:  Chloramphenicol, E. Erythromycin, S:  Streptomycin, SXT: Trimethoprim,   R= resistance,              S =   sensitive

Table 4: Results of Salmonella typhimurium colony count from fecal samples of chicken in non-treated and treated experimental groups.

Table 5: Results of Salmonella typhimurium colony count from internal organs of chicken in non-treated and treated experimental groups. (Total Salmonella Counts: Log₁₀ CFU/g).

   Means (± S.E) with different superscript (a, b, c, d) within the same column are significantly different at p<0.05

Table 6: The hematological picture on broiler chicks experimentally infected with salmonella typhimurium and infected treated groups.

Means (± S.E) with different superscript (a, b, c, d) within the same column are significantly different at p<0.05

Table 7: Some biochemical tests on broiler chicks experimentally infected with salmonella and infected treated groups

    Means (± S.E) with different superscript (a, b, c, d) within the same column are significantly different at p<0.05

Table 8: Protienogram on broiler chicks experimentally infected with salmonella and infected treated groups

     Means (± S.E) with different superscript (a, b, c, d) within the same column are significantly different at p<0.05

Table 9: Haemagglutination inhibition test in different groups

Means (± S.E) with different superscript (a, b, c, d) within the same column are significantly different at p<0.05.

Figures:

DISCUSSION

Salmonellosis is considered one of the anthropozoonotic diseases of a serious medical problem and raises great concern in the food industry. Poultry is the most potential source of Salmonella food poisoning in man (Ashton, 1990).

The gross lesions in the liver showed variable changes including friable and bronze discoloration with white necrotic foci. Lungs were congested and pneumonic. Spleen was enlarged with discoloration. Intestine showed various grades of enteritis with mucous in the lumen. These findings were supported by Islam et al. (2006);Msoffe et al. (2006) and Deshmukh et al. (2007).

Regarding Salmonella organisms isolation there were, different cultural characteristics in different media. These were red / black center colony on XLD, pink white color colonies in BGA, whitish or slightly grayish color colonies in SS agar, red slant/ yellow butt with H₂S production in TSI agar, pale color colonies in MacConkey agar, well defined glistening colonies in blood agar and pinkish colonies in EMB agar. These results were supported by Rybolt et al. (2005). From pure culture of different media, Gram’s staining was performed to observe Salmonella organism. Under compound light microscope the organism was identified as Gram-negative, rod shaped and arranged in single bacilli. Chains of more than two bacilli were normally absent. Freeman (1985) described the morphology of the isolated Salmonella bacteria as small rod shaped, gram negative, single or paired in arrangement by Gram’s staining which supported the findings of the present study. Similar morphological findings of Salmonella were also supported by other investigators(Saha et al., 2012). Different biochemical tests were used for identification of Salmonella organism in this study. All Salmonella were MR positive and VP and indole negative. In the dulcitol fermentation test, Salmonellae fermented dextrose (Haider et al., 2003).

In the present study, the prevalence of Salmonella typhimurium based on bacteriological and biochemical characters was found to be 6% (3/50) in diseased broiler chickens and 3.33%(10/300) from organs of dead chickens which was much lesser than those reported by Zivkovic et al. (1997). The percentage isolation of salmonella typhimurium from chickens generally in this study is more or less similar to that published by several previous authors as Badr and Abd El Monaem (2008) and Muhammad et al. (2010) which ranged from 1.7% to 28.6%. Kobayashi et al. (2006) isolated 19 strain of salmonella typhimurium from 328 cloacal swabs of birds.

The level of contamination of liver and spleen (5%) was significantly lower than the level (23.11%) as reported by Zivkovic et al. (1997) but higher than the reports (2.80%) by Ozbey and Ertas (2006). The level of Salmonella isolation in broilers evaluated in this study may be attributed to horizontal and/or vertical transmission of Salmonella to the chicks. The horizontal transmission of Salmonellae can be mediated by mechanisms including direct bird-to-bird contact, ingestion of contaminated feeds, water or litter, or using contaminated equipment, while vertical transmission to the progeny of infected breeder flocks can result from internal or external contamination of eggs (Gast, 2003). This investigation highlights the importance of isolation of Salmonella from broilers as it was reported that slaughtering broiler flocks infected with Salmonella can lead to contamination both of the carcasses and the slaughter line (Corry et al., 2002 and Olsen et al., 2003).

The obtained results of antimicrobial sensitivity test revealed that the thymus vulgarisextract was found to be effective against isolated microorganisms in this study. Notably, the isolated bacteria were more susceptible to the oily thymus vulgar is extract as compared to the standard antibiotic discs except for ciprofloxacin and enrofloxacin. The isolates showed highest antibiotic sensitivity tociprofloxacin and enrofloxacin (100.00% sensitivity) which was incorrelation to the reports of Zahrei et al. (2005). The nexthighest sensitivity was found to chloramphenicol asreported by Hui and Das (2001).

The in vitro activity of thyme oil against Salmonella Typhimurium was reported to range from 0.45 to >20 μL/mL (Burt, 2004). Generally, essential oils have been effective at penetrating the outer membrane of salmonella and E. coli which is necessary for bactericidal activity (Helander et al., 1998). Thymol (5-methyl-1-2-isopropyl phenol) and carvacrol (5-isopropyl-2-methyl phenol) are the main phenolic components in Thymus vulgaris (Masada, 1976) and antibacterial activity of thyme extract, oil, and the major componentsagainst of Clostridium botulinum, Clostridium perfringens, Bacillus subtilis, S. sonnei, E. coli, H. pylori, S. typhimurium, S. sonnei, Bacillus cereus, L monocytogenes, C. jejuni and S. enteric were reported in previous literatures (Nevas et al., 2004 and Thakare, 2004). Bacillus cereus, L monocytogenes, C. jejuni and S. enteric reported in previous literatures (Nevas et al., 2004 and Thakare, 2004).

Colonization of salmonellae in different organs was supported in this study byisolating salmonellae from the liver, spleen, lung and cecal pouches at 7 DPI which indicated that the chicks were infected. Thececal pouch was identified as an ideal organ for Salmonella isolation during acute andchronic infection, compared with other organs, and it poses a potential source forenvironmental contamination (Dhillon et al., 2001 and Osman et al., 2010). In the intestinal tract itself, the cecal contents are the major site for recovery of serotype s. Typhimurium in the cecum of 1-week-old chicks (Sivula et al., 2008).

The results of Salmonella typhimurium colony count from fecal and internal organs samples of chicken in non-treated and treated experimental groups showed the decreased number of colony count after treatment by thyme extract and ciprofloxacin. These results indicated bactericidal effect of thyme on salmonella. These results agreed with Hoffman-Pennesi and Wu (2010).

Concerning to leukogram, there was leukocytosis and lymphocytosis when prebiotic (G2) was used. Ihis may be attributed to immunostimulatory and immunomodulatory effect of prebiotic. The obtained results are in agreement with EL-Boshy     et al. (2008) who reported that the absolute lymphocyte count (ALC) values increased in supplemented group with prebiotic indicating better immune response that could be attributed to immunostimulatory effect of prebiotics. Herbs that are rich in such flavonoids as thyme (Thymusvulgaris) extend the activity of vitamin C act as antioxidants and may therefore enhance the immune function (Cook and Samman, 1996). Meanwhile after Salmonella challenge there was a significant increase in total leukocyte count and heterophil count compared to control group at 3 and 7 days post infection. The obtained results are similar to those reported by Ogunleye et al. (2009) and Salim et al. (2011).

Regarding the results of serum biochemical analysis, the obtained findings displayed that Salmonella typhimurium 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). 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) and Schimidt et al. (2007) reported that the activity of hepatic enzymes, as ALT and GGT, increase when there are hepaticlesion due to overflowing of these enzyme currents of the compromising of the hepatocyte’s membrane.

The infected birds treated with ciprofloxacin ad thymus vulgaris displayed a significant decrease in serum AST and ALT values at the 7^th and the 14^th days post treatment as compared to the infected untreated group. These findings may be attributed to the antioxidant effect of thyme as stated by Cook and Samman (1996).

On the other hand there was a significant increase of uric acid and creatinine after challenge with Salmonella typhimurium as a result of renal damage. Similar results recorded by Guo et al. (2003) showed significant increase in serum uric acid and creatinine during the experimental period in Salmonella infected group.

Concerning serum proteins, there was significant decrease in serum total protein and albumin levels and significant increase in globulin (Table 6) as compared with control group at the 3^rd day post infection in all infected groups when compared to the control group. These results indicated hepatic damage because liver is responsible for the production of a great proportion of plasma protein (Coles, 1986). The ciprofloxacin treated birds showed a non-significant change in serum total protein and albumin at the 7^thday and the 14^thpost treatments as compared to infected non treated group. These findings indicated improvement of hepatic function. The infected birds and  treated with oily thymus vulgaries extract showed non-significant changes in total proteins and albumin at the 7^th and the 14^th post treatment as compared  to control group and infected non medicated group. These results indicate improvements in hepatic functions because of hepatoprotective effect of thymus extract owing to the antioxidant effect of its higher contents of phytophenolic compounds as previously discussed. However, albumin serum concentrations showed relevant alterations only in the 7^thday of the G3. Albumin, which is synthesized only in the liver mainly responsible for maintaining the oncotic blood pressure. It may occur hypoalbuminemia in cases of hepatic insufficiency, malnutrition and gastrointestinal disturbances within other conditions (Meyer et al., 1995)

Gel electrophoresis showed that significant increase in serum gamma globulins of thymus group compared to the control group. Thyme is characterized by its immunity through increasing gamma globulins due to effect of thyme on immunity. While there was significant increase in serum alpha and gamma globulins of infected non treated group and infected treated groups with thymus extract compared to control group. These results agreed with Mansoub and Nezhady (2011). An elevation in the alpha and gamma globulins usually indicates activation of the immune system and is most often due to infection or inflammatory diseases (Butler, 1983).

Regarding to antibody titer against Newcastle ND, there was a significant increase in antibody titer against ND as a result of administration of thyme group and in group 4 which is infected and treated by thyme extract. These results agreed with Mansoub and Nezhady (2011).

Conclusion

The obtained results concluded that thyme has the ability to reduce the adverse effect of Salmonella typhimurium infection in broiler chicks so it can be agood alternative for commercial antibiotics. Thyme is considered immunostimulant due to stimulation of immune system by increasing lymphocyte cell, antibody titer, alpha, and gamma globulin. On the other hand, there is a microbial resistance danger because of antibiotics overuse. We need to use such natural products to improve the performance of poultry.

REFERENCES

Abdulkarimi, R.; Daneshyar, M. and Aghazadeh, A.M. (2011): Thyme (Thymus vulgaris) extract consumption darkens liver, lowers blood cholesterol, proportional liver and abdominal fat weights in broiler chickens. Italian Journal of Animal Science, 10:20.

Abu-Darwish, M.S. and Abu-Dieyeh, Z.H.M. (2009): Essential Oil Content and Heavy Metals Composition of Thymus vulgaris Cultivated in Various Climatic Regions of Jordan. Int. J. Agric. Biol., 11(1): 59-63.

Aksit, M.E.; Goksoy, F.; Ozdemir, D. and Ozdogan, M. (2006): The impact of organic acid and essential oil supplementations to diets on microbiological quality of chicken carcasses Arch. Geflugleked, 70:168-173.

Ashton, W.L.G. (1990): Enterobacteriaceace in PoultryDisease. 3^rd ed. El. Baunere, pp: 11-41.

Badr, J.M. and Abd El Monaem, H. (2008): Antigenic variations between different Salmonella serotypes isolated from chickens. Assiut Vet. Med. J. 54, 373–388.

Biondi, D.; Cianci, P.; Geraci, C.; Ruberto, G. and Piattelli, M. (1993): Antimicrobial activity and chemical composition of essential oils from Sicilian aromatic plants. Flavour Fragrance J. 8: 331-337.

 Burt, S. (2004): Essential oils: Their antibacterial properties and potential applications in foods—A review. Int. J. Food Microbiol. 94:223–253.

Butler, J.E. (1983): Bovine immunoglobulins: an augmented review. Vet. Immunol. Immunopathol. 4(1-2):143-152. 

Campbell, T.W. and Coles, E.H. (1986): Avian Clinical Pathology In: Coles, E., Vet. Clin. Pathol., 4th Ed., pp: 279-301.

Caraway, W.T. (1963): Standard Methods of Clinical Chemistry. Edited by Seligsol, D. Academic Press, New York and London, 4.

Cardoso, T.G. and Carvalho, V.M. (2006): Toxinfecção por Salmonella spp. Revista do Instituto de Ciências da Saude, 24(2): 95-101.

Coles, H.E. (1986): Veterinary Clinical Pathology. 4^th Ed. W.B. Saunders Co., Philadelphia.

Cook, N.C. and Samman, S. (1996): Flavonoids-chemistry, Metabolism, Cardioprotective Effects, and Dietary Sources. J. Nutr. Bioche,7: 66-76.

Corry, J.E.L.; Allen, V.M.; Hudson, W.R.; Breslin, M.F. and Davies, R.H. (2002): Sources of Salmonella on broiler carcasses during transportation and processing: modes of contamination and methods of control. Journal of Applied Microbiology, 92: 424-432.

Demir, E.; Kilinc, K.; Yildirim, Y.; Dincer, F. and Eseceli, H. (2008): Comparative effects of mint, sage, thyme and flavomycin in wheat-based broiler diets. Archiva Zootechnica 11(3): 54-63.

Deshmukh, S.; Asrani, R.K.; Ledoux, D.R.; Rottinghaus, G.E.; Bermudez, A.J. and Gupta, V.K. (2007): Pathologic changes in extrahepatic organs and agglutinin response to Salmonella Gallinarum infection in Japanese quail fed Fusarium verticillioides culture material containing known levels of fumonisin B1. Avian Dis. 51: 705-712.

Dhillon, A.S.; Shivaprasad, H.L.; Roy, T.P.; Alisantosa, B.; Schaberq, D.; Bandli, D.; Fan, M. and Chen, J. (2001): Studies on antimicrobial activity of extracts from thyme. Wei Sheng Wu Xue Bao, 41:499-504.

 Doumas, B.T. (1971): "Standard Method for Clinical Chemistry", vol .7, Academic Press, New York. Clinical Chemistry, 21:10.Acta, 31-78.

EL-Boshy, M.E.; EL-Ashram, A.M.M. and Abd El-Ghany, N.A. (2008): Effect of dietary beta1, 3 glucan on immunomodulation on diseased Orechromis niloticus experimentally infected with aflatoxin B1. 8^th International Symposium on Tilapia in Aquaculture. Pp. 1109-1127.

Erdem, B.; Erics, S.; Hascelik, G.; Gur, D. and Aysev, A.D. (2005): Antimicrobial resistance of Salmonella enteric group C strains isolated from humans in Turkey,2000-2002. Int.J. Antimicrob. Agents,26:33-37.

Freeman, B.A. (1985): Burrow's Text Book of Microbiology. 2^nd ed. W.R. Saunders Company, London, UK. 372-472.

Frink, C.R.; Waggoner, P.E. and Ausubel, H. (1999): Nitrogen fertilizer: retrospect and prospect. Proc. Natl. Acad. Sci., USA. 96: 1175-1180

Gast, R.K. (2003): Paratyphoid infections. In: Diseases of poultry, 11^th ed. (Eds Saif, Y.). Iowa State University Press, Ames. 583-613.

Grimont, P. and Weill, F. (2007): Antigenic Formulae of the Salmonella Serovars, 9^th ed Paris: World Health Organization Collaborating Centre for Reference and Research on Salmonella,Pasteur Institute.

Guo, Y.; Ali, R.A. and Qureshi, M.A. (2003): The influence of β-glucan on immune responses in broiler chicks. Immunopharmacol. Immunotoxicol. 25: 461–472.

Haider, M.G.; Hossain, M.G.; Hossain, M.S.; Chowdhury, E.H.; Das, P.M. and Hossain, M.M. (2003): Isolation and characterization of enterobacteria associated with health and disease in Sonali chickens. Bang. J. Vet. Med. 2: 15-21.

Helander, I.M.; Alakomi, H.; Latva-Kala, K.; Mattila-Sandholm, T.; Pol, I.; Smid, E.J.; Gorris, L.G.M. and Von Wright, A. (1998): Characterization of the action of selected essential components on gram-negative bacteria. J. Agric. Food Chem. 46: 3590–3595.

Henry, R.J. (1964): "Clinical Chemistry, Principles and Techniques". New York: Harper and Row Publishers, p. 181.

Hoffman-Pennesi, D. and Wu, C. (2010): The Effect of Thymol and Thyme Oil Feed Supplementation on Growth Performance, Serum Antioxidant Levels, and Cecal Salmonella Population in Broilers. Journal of Applied Poultry Research, 19, 432-443.

Hui, A.K. and Das, R. (2001): Studies on isolation, serotyping and antibiotic sensitivity of Salmonella isolated from ducks. Indian Vet. J., 78(11): 1058-1059.

Islam, M.M.; Hossain, M.M.; Haider, M.G.; Chowdhury, E.H. and Kamruzzaman, M. (2006): Seroprevalence and pathological study of Salmonella infections in layer chickens and isolation of causal agents: In Proceedings of the 5^th International Poultry show and seminar from 01-03 march 2007, held in Bangladesh China Friendship Conference Centre (BCFCC), Sher-e-Bangla Nagar, Dhaka, Bangladesh. 9-15.

Jain, N.C. (2000): Schalm's veterinary hematology. 8th Ed. Lea and Febiger, Philadelphia,U.S.A.

Kobayashi, H.; Kanazaki, M.; Shimizu, Y.; Nakajima, H.; Minara Khatun, M.; Hata, E. and Kubo, M. (2006): Salmonella isolates fromcloacal swabs and footpads of wild birds in the immediate environment of Tokyo Bay. Note Bacteriology, pp309-311.

Mansoub, N.H. and Nezhady, M.A. (2011): Effect of garlic, thyme and yogurt compared to antibiotics on performance, immunity and some bloodparameters of broiler chickens. Indian Journal of Animal Sciences. 81 (12): 1197–1200.

Markovicva, R.; Šefera, D.; Krsticvb, M. and Petrujkicva, B. (2009): Effect of different growth promoters on broiler performance and gut morphology. Arch. Med. Vet. 41: 163-169.

Masada, Y. (1976): Analysis of essential oils by gas chromatography and mass spectrometry. John Wiley and Sons Inc., New York NY, pp.: 334.

Meyer, D.J.; Coles, E.H. and Rich, L.J. (1995): Medicina de Laboratório Veterinária. São Paulo: Interpretação e Diagnóstico. Roca, 307.

Miceli, A.; Tommasi, L.; Negro, C. and De Leo, P. (2002): Composizione e variabilita` dell’olio essenziale di T. capitatus. In: Proceeding of LXV Congress of SIFV, Riva del garda (TN), 20-23 September

Mikaili, P.; Mohammad Nezhady, M.A.; Shayegh, J. and Asghari, M.H. (2010): International Journal of  Academic Research, 2(6): Part II

Msoffe, P.L.M.; Minga, U.M.; Mtambo, M.M.A.; Gwakisa, P.S. and Olsen, J.E. (2006): Differences in resistance to Salmonella Enterica serovar Gallinarum infection among indigenous local chicken ecotypes in Tanzania. Avian Path. 35: 270-276.

Muhammad, M.; Lawal, U.M.; Abdul-Ganiyu, A.; Aliyu, U.M.; Samuel, A. and Lisa, B. (2010): Prevalence of Salmonella associated with chick mortality at hatching and their susceptibility to antimicrobial agents. Veterinary Microbiology 140, 131-135.

National disease surveillance center (2004): Annual Report 2003.

NCCLS (National Committee for Clinical Laboratory Standards) (2002): Performance standards for antimicrobial discs and dilution susceptibility for bacteria isolated from animals, Approved Standards-Second Edition, (22): 1-52.

Nevas, M.; Korhonen, A.R.; Lindstrom, M.; Turkki, P. and Korkeala, H. (2004): Antibacterial efficiency of Finnish spice essential oils against pathogenic and spoilage bacteria, Journal of Food Protection, 67: 199-202.

Ogunleye1, A.O.; Ajuwape, A.T.P. and Adetosoyel, A.I. (2009): Hematological Changes in Salmonella Paratyphi A Infected Pullets. Revue Élev. Méd. vét. Pays trop., 62 (1): 23-26.

Olsen, J.E.; Brown, D.J.; Madsen, M. and Bisgaard, M. (2003): Cross contamination on a broiler slaughterhouse line demonstrated by use of epidemiological markers. Journal of Applied Microbiology. 94: 826-835

Osman, K.M.; Ihab, M.I.M.; Ashgan, M.M. Youssef; Mona M. Aly; Moustafa, R. and Alwathnani, H.A. (2010): Pathogenicity of Some Avian Salmonella Serovars in Two Different Animal Models: SPF Chickens and BALB/c Mice. Environ. We Int. J. Sci. Tech. 5: 65-78.

Ozbey, G. and Ertas, H.B. (2006): Salmonella spp. iaolation from chicken samples and identification by polymerase chain reaction.  Bulgerian. J. Vet. Med., 9(1): 67-73.

Quinn, P.J.; Markery, B.K.; Carter, M.E.; Donnelly, W.J. and Leonard, F.C. (2002): Veterinary Microbiology and Microbial Diseases. Block well Science Ltd. 1st Published.

Reitman, S. and Frankel, S. (1957): Colorimetric determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase. Am. J. Clin. Path. 28: 56-58.

Rybolt, M.L.; Wills, R.W. and Bailey, R.H. (2005): Use of secondary enrichment for isolation of Salmonella from naturally contaminated environmental samples. Poult. Sci. 84:        992-997.

Saha, A.K.; Sufian, M.A; Hossain, M.I. and Hossain, M.M. (2012): Salmonellosis in layer chickens: pathological features and isolation of bacteria from ovaries and inner content of laid eggs. J. Bangladesh Agril. Univ. 10(1): 61–67.

 Salim, H.A.; Abd-Allah, O.A. b and Fararh, K.M. (2011): Clinicopathological study on the effect of beta-glucan on hematological and immunological and biochemical changes in broiler chicks. Benha veterinary medical journal, 22 (2): 68-77.

Schimidt, E.MS.; Locatelli-Dittrich, R.; Santin, E. and Paulillo, A.C. (2007): Clinical pathology in poultry – A tool to improve poultry health – a review. Archives of Veterinary Science, 12:    9-20.

Schlundt, J.; Toyofuku, H.; Jansen, J. and Herbst, S.A. (2004): Emerging food-borne zoonoses. Rev. Sci. Tech.; 23: 513-533.

Silva, A.M.; Bambirra, E.A.; Oliveira, A.L.; Souza, P.P.; Gomes, D.A.; Vieira, E.C. and Nicoli, J.R. (1999): Protective effect of bifidus milk on the experimental infection with Salmonella enteritidis subsp. Typhimurium in conventional and gnotobiotic mice. J. Appl. Microbiol., 86: 331-6.

Sivula, C.P.; Bogomolnaya, L.M. and Andrews-Polymenis, H.L. (2008): A comparison of cecal colonization of Salmonella enterica serotype Typhimurium in white leghorn chicks and Salmonella- resistant mice. BMC Microbiology. 8:182.

Thakare, M. (2004): Pharmacological screening of some medicinal plants as antimicrobial and feed additive. M. S. Thesis, Virginia Polytechnic Institute and State University, Blacksburg, Virginia USA.

Thrall, M.A.; Baker, D.C.; Campbell, T.W.; DeNicola, D.; Fettman, M.J.; Lassen, E.D. and Rebar, A. (2004):   Clinical Chemistry of Birds. In: Veterinary Haematology and Clinical Chemistry, Lippincot Williams and Wilkins, U.S.A., P: 479-492.

Waltman, W.D. (1999): Methods for Isolating Salmonellae from Poultry and the Poultry Environment in: Salmonella enterica Serovar Enteritidis in Humans and Animals; epidemiology, pathogenesis, and control. (Eds. Saeed A. M.; Gast R. K.; Potter M. A. and Wall P. G.). Iowa State University Press, Ames, Iowa, USA. 419-432.

Young, D.; Pestaner, L. and Giberman, V. (1975): Clinical Chemistry, 21:10.

Zahrei, S.T.; Mahzounish, M. and Saeed, Z. (2005): The isolation of antibiotic resistant Salmonella from intestine and liver of poultry in Shiraz province of Iran. Int. J. Poult. Sci., 4(5):     320-322.

Zhang, F.; Chen, B.; Xiao, S. and Yao, S.Z. (2005): Optimization and comparison of different extraction techniques for sanguinarine and chelerythrine in fruits of Macleaya cordata (Willd) R. British Separation Purification Technol. 42:283-290.

Zivkovic, J.; Jacsic, S. and Miolovic, B. (1997): Salmonella serovars in chicken meat and chicken meat products in Zagrep, Croatia. Vet. Archive, 67(4): 169-175.