COMPARISON OF EFFICACIES OF FOSFOMYCIN, THIAMPHENICOL, NIGELLA SATIVA OIL AND ITS COMBINATION AGAINST ESCHERICHIA COLI INFECTION IN CHICKENS

ABEER M. RADI^* and NESREEN A. IBRAHEEM^**

^* Department of Pharmacology, Faculty of Veterinary Medicine, Beni-Suef University

^**Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Beni-Suef University

Email:rouqaa2007@yahoo.com

INTRODUCTION

Escherichia coli is one of the most important and frequently encountered bacterial avian pathogens causing a wide variety of disease syndrome in birds causing up to 30% of poultry mortality (Geornaras    et al., 2001), also it is responsible for severe economic losses for the world's poultry industries (Barnes et al., 2008).

Avian pathogenic Escherichia coli induces different syndromes in poultry including systemic and localized infections such as  respiratory colibacillosis, acute colisepticemia with characteristics fibrinous lesions (air saculitis, perihepatitis, and pericarditis), salpengitis, yolk sac infection, and swollen-head syndrome (Dho-Moulin and Fairbrother, 1999). The infection is generally initiated or enhanced by predisposing agents, such as mycoplasmal or viral infections, and environmental factors. Chickens of all ages are susceptible to collibacillosis (Barnes et al., 2003).

Fosfomycin is a broad-spectrum bactericidal agent that attacks bacteria by inhibiting cell wall synthesis (Dàmaso, 1990). This antibiotic posses high activity in vitro against a wide range of Gram- positive bacteria such as Streptococcus sp., S. aureus and some Gram- negative bacteria such as Pseudomonas aerugenosa and E.coli (Tessier& Quentin, 1997; Grif et al., 2001). It is used in chickens for the treatment of infection caused by E.coli and Salmonella sp. (Prescott, 2000). Pharmacokinetics studies in broiler chickens have shown that fosfomycin sodium salt is a product very soluble in water, with low protein binding, and low molecular weight, which hinders its diffusion within tissues such as muscle or fat, reaching a high concentration in the kidney (Aramayona et al., 1997).

Thiamphenicol is a broad-spectrum bacteriostatic antibiotic, inhibiting the bacterial protein synthesis at the ribosome (Cannon et al., 1990). It is a structural analogues of chloramphenicol with lower toxicity, in addition to it has a greater in vivo activity against pathogenic bacteria than other structural analogues and it's also active against some bacteria that are resistant to chloramphenicol. It has been approved in European Union for use in cattle, sheep, pigs and chickens (Giorgi et al., 2000).

The antimicrobial agents of plant origin such as essential oils, plants extracts and complete plant substances, have gathered significant consciousness as alternatives to the traditional antibacterial feed additives. Extracts of Nigella sativa oil have shown promising effects against bacteria, fungi, viruses, parasitics and worms. Seed extracts of Nigella sativa were found to inhibit the growth of Escherichia coli, Bacillus subtilis and streptococcus faecalis (Hala      et al., 2006).

Supplementing broiler diets with Nigella sativa seed, oil or meal improve growth performance, biochemical and hematological response and mortality rate (Nofal et al., 2006). Feeding different forms and levels of NS has been reported to possess anti stress activity (Tolba and Hassan, 2003), antimicrobial activity (Nasir and Grashorn, 2006) and has a stimulatory effect on the immune system (Tolba et al., 2005).

This present study was designed to compare the efficacy of fosfomycin and thiamphenicol as antibacterial agents and their effects in combination with Nigella sativa oil for treatment of experimental infections with E. coli O₁₁₁. Also, the effect of Nigella sativa oil on the immune response was investigated.

MATERIALS and METHODS

Birds

A total of 126 Hubbard broiler chicks, one-day old were floor reared under suitable conditions of husbandry. Commercial diets (antibacterial free) and water were provided ad libitum. The chicks were allowed a 5 days acclimatization period prior to the study.

Bacteria

The E. coli strain O₁₁₁ was kindly supplied by Dokki animal health research institute. The bacteria were grown in BHI broth (Oxoid) for 24 hours at 37˚C and then on BHI agar (Oxoid) for 24 hours at 37˚C. To prepare the inoculums, the colonies were suspended in sterile saline and compared with McFarland 0.5 to obtain a concentration of 1.8 ×10⁸CFU/ml.

Drugs:

Disodium fosfomycin, Thiamphenicol and its solvent (dimethylacetamide) were provided as pure powdered from (Pharma Swede Company, Egypt) and Nigella sativa oil was obtained from (Royal herbs, ltd company, Egypt).

Experimental design:

The 126 one-day old chicks were divided into seven groups (18 chicks each). At the 5^th day of age the birds in 5^th, 6^th, 7^th groups were orally administered with Nigella sativa oil daily till the end of the experiment (35 day of age). At the 12^th day of age the chicks were experimentally infected with E. coli O₁₁₁ (1ml of 1.8×10⁸ CFU/ bird) and the treatment by fosfomycin and thiamphenicol began after 6 hours from infection and lasted for 5 successive days (Rawiwet et al., 2010) as the following.

The 1^st group was left as control (non infected, non medicated), the 2^nd group was infected with E. coli O₁₁₁ and not medicated, the 3^rd group was infected with E. coli O₁₁₁ and treated with disodium fosfomycin in a dose of 40mg/kg b.wt (Gutierrez      et al., 2010) in drinking water, the 4^th group was infected with E. coli O₁₁₁ and treated with thiamphenicol in a dose of 30mg/kg b.wt (Switala     et al., 2007) in drinking water.

While the 5^th group was infected with E. coli O₁₁₁ and orally administrated with Nigella sativa oil in a dose of 0.025 ml/bird (by using plastic tube directly into the crop) (Hodžic et al., 2012). The 6^th group was infected with E. coli O₁₁₁ and treated with disodium fosfomycin in a dose of 40mg/kg b.wt in drinking water and orally administered with Nigella sativa oil in a dose of 0.025 ml/bird. The 7^th group was infected with E. coli O₁₁₁ and treated with thiamphenicol in a dose of 30mg/kg b.wt in drinking water and orally treated with Nigella sativa oil in a dose of 0.025 ml/bird.

The chicks were examined daily for clinical signs of the disease and mortality. The body weight of all chicken were determined at the beginning of the experiment and daily for 5 days during administration of the tested drugs then weekly till the end of experiment (35 day), also feed and water consumption were determined during the 5 days of medication to determine the feed conversion rate (FCR=feed intake (g)/ average weight gain (g) ) and also Average daily gain (ADG) was determined and calculated from the formula (ADG= weight gain (g)/ days).

Blood sample were taken from 5 birds from each group (from wing vein) in the first day of infection and medication and after 2 hour from administration of the drugs and/or oils and daily during the 5 days of medication for determination of the antibacterial activity of tested drugs  as diameter zone of inhibition against E coli O₁₁₁. Also, the blood samples were collected from chicken of each group at 0, 7^th, 15^th and 21^st day of infection for detection of the immune response against E. coli O₁₁₁. The blood samples allowed to clot at room temperature for 30min., after which the sample were centrifuged at 3000 rpm for 15 min. The separated serum was decanted and immediately frozen at -20˚C until use.

Efficacy criteria:

The air sac, pericardial and perihepatic lesions of colisepticemia in each of the three sacrificed birds from each group at the 2^nd, 5^th, 8^th and 10^th day of the infection were scored. According to Charleston et al., 1998, the air sac lesions of colisepticemia were scored as follows: 0: no lesions, 1: cloudness of air sacs, 2: air sac membranes are thickened, 3: "meaty" appearance of membranes with large accumulation of cheesy exudates confined to one air sac and 4: lesions with the same score as score 3 but with lesion in two or more air sacs while the pericardial lesions were scored as follows: 0: no lesions, 1: excessive clear or cloudy fluid in the pericardium, 2: extensive fibrination in pericardial cavity. For perihepatic lesions, 0: no visible lesion, 1: definite fibrination on the surface of the liver, 2: extensive fibrination, adhesions, liver swelling and necrosis.

To re-isolate the challenge E.coli O₁₁₁, swabs from the liver, spleen and heart were cultured on MacConkey agar (Oxoid) at 37˚Cfor 24 hours.

Antibacterial activity of the tested drugs on E.coli O₁₁₁:

In vitro antibacterial activity was determined by the agar well-diffusion method (Mukherjee et al., 1995). The E. coli O₁₁₁ was cultured on Muller Hinton agar for 24h at 37˚C then the bacterial cells were harvested and re-suspended in saline to make a suspension of 10⁵ CFU/ ml and used for assay. The bacterial suspension (10⁵ CFU/ ml) was mixed with Muller Hinton agar medium then the media was transferred to sterile Petri-plates (25ml/plate) and allowed to solidify. About 100µl of serum sample (which collected from chicken after 2h from administration of drugs) was placed in the wells and allowed to diffuse, then the plates were incubated at 37˚C for 24h. The antibacterial activity of the tested drugs was determined by measuring the diameter of the inhibition zones. The assay was performed in triplicate.

Immune-responsiveness against E. coli O₁₁₁:

The antibody titers against E. coli serotype O₁₁₁ were determined in serum samples collected from each group on 0, 7, 15 and 21 days post infection by using Enzyme linked Immnosorbent Assay (ELISA) according to Briggs and Skeeles (1984).

Statistical analysis

ANOVA, student –t test was used for statistical comparison of the body weight, food conversion rate and average daily gain. The lesion scores were analyzed by Mann-Whitney U-test. The ELISA data were analyzed by using the General Linear Models. SPSS for windows was used for statistical analysis.

RESULT

Efficacy of fosfomycin, thiamphenicol and Nigella sativa oil against E. coli O₁₁₁ infection:

Clinical signs were absent from control (non infected non medicated) group. The chicken infected with      E. coli O₁₁₁and not treated were depressed, ruffled feathers, showed signs of anorexia, diarrhea and respiratory signs without mortalities. The chicken infected with E. coli and treated with fosfomycin, thiamphenicol and Nigella sativa oil showed less signs than infected group.

The post mortem pathology of the sacrificed chickens revealed that the infected non treated group individuals had severe lesions airsacculitis of, perihepatitis, pericarditis, while the lesions were less severe in chicken of the treated groups. The results of the lesion scores were recorded in table (1), which showed that there was significant decrease (p<0.05) in lesion scores of treated group.

Table 1: Mean macroscopic lesion scores in sacrificed chicken in infected non- treated and treated groups.

1^st group was Control group, 2^nd group was Infected group, 3^rd group was Fosfomycin treated group, 4^th  group was thiamphenicol treated group, 5^th group was Nigella sativa oil treated group, 6^th group was Fosfomycin + Nigella sativa oil treated group and 7^th  group was Thiamphenicol + Nigella sativa oil treated group

a Group that significantly different (P < 0.05) from the 1^st group

b Group that significantly different (P < 0.05) from the 2^nd  group

The E. coli O₁₁₁was re-isolated in pure culture from sacrificed chicken of infected non treated group until the 10^th day of infection while in the treated groups the re-isolation of the E. coli O₁₁₁was negative from the 8^th day of infection.

Performance:

The mean body weight and feed conversion rate at the treatment period were determined in all groups (table, 2). Also the mean body weight and the average daily gain were determined at the end of experiment, and illustrated in (table, 3). The results showed that the groups infected with E. coli O₁₁₁and treated with fosfomycin, thiamphenicol and/or Nigella sativa oil showed improvement in body weight than infected non treated group, also chickens infected with E. coli O₁₁₁and non medicated showed a decrease in food consumption and feed conversion ratio (FCR) when compared with control group, while infected chicken medicated with fosfomycin, thiamphenicol and/or Nigella sativa oil showed significant increase in FCR when compared with infected group.

The ADG were significantly increased in all infected treated groups, especially groups treated with fosfomycin and thiamphenicol in combination with Nigella sativa oil than infected non treated group.

Table 2: The mean body weight (MBW; g/chick) and feed conversion rate (FCR) at the treatment period in all groups.

Groups that significantly different from the 2^nd group,          ٭p <0.05                  ٭٭ p <0.01             ٭٭٭p <0.001

Table 3: The mean body weight and the average daily gain at the start of experiment and at the end of experiment in all groups.

Groups that significantly different from the 2^nd group,          ٭p <0.05                 ٭٭ p <0.01                       

Antibacterial activity of the tested drugs against E coli O₁₁₁:

Antibacterial activity of fosfomycin, thiamphenicol and Nigella sativa oil against E coli O₁₁₁ measured as diameter zone of inhibition (mm) and illustrated in table (4). The result showed that the administration of Nigella sativa oil with fosfomycin or thiamphenicol increase their antimicrobial activity, the serum samples of the group treated with fosfomycin and Nigella sativa oil showed the highest zone of inhibition than other groups.

Table 4: Antibacterial activity of fosfomycin, thiamphenicol and Nigella sativa oil against E. coli O₁₁₁.

Immune-responsiveness against E. coli O₁₁₁:

The data showed significantly increase in antibody titer (p < 0.05) for E. coli O₁₁₁ after two weeks of infection in all chicken groups which infected or infected and received antibacterials alone or in combination with Nigella Sativa oil  than those of the control group (Table, 5).

The 3^rd, 5^th group, 6^th group and 7^th group revealed significant increase (P < 0.05) in antibody titer against E. coli O₁₁₁ in comparison with infected group after two weeks of infection. The important comparison for antibody titer in groups which received Nigella Sativa oil either with fosfomycin or thiamphenicol showed that the level of antibody production significantly elevated after 15 days and continued after the 21 days of infection.

Table 5:Mean log₁₀ antibody titer against E. coli serotype O₁₁₁ in all groups at 0, 1^st, 2^nd and 3^rd week post infection.

a Group that significantly different (p < 0.05) from the 1^st group

b Group that significantly different (p < 0.05) from the 2^nd  group

Figure 1:Mean log₁₀ antibody titer against E.coli serotype O₁₁₁ in all groups at 0, 1^st, 2^nd and 3^rd week post infection.

DISCUSSION

A major problem in antimicrobial chemotherapy is the increasing occurrence of resistance to antibiotics, which lead to insufficiency of antimicrobial treatment (Schelz et al., 2006), so combination of different antimicrobials may result in a predictable therapeutic result as to increase the therapeutic efficacy or decrease the toxicity among the administrated drugs (Hurry et al., 1998). Therefore the aim of this work is to compare the efficacy of fosfomycin, thiamphenicol alone or in combination with Nigella Sativa oil against experimental infection of broiler chickens with E. coli O₁₁₁ and study their effect on immune response.

In the present study, the experimental infection of broiler chickens with E. coli O₁₁₁ cause severe clinical signs (depression, decrease food intake, respiratory sings and diarrhea) with gross lesions (airsacculitis and pericarditis) in non medicated group, this was similar to those reported by (Nakamura et al., 1992; Mogenet et al., 1997). The clinical signs and lesion scores were less in chicken treated with fosfomycin, thiamphenicol alone or in combination with Nigella sativa oil, these results were similar to those reported by (Fernandez et al., 2002) who proved that the treatment with calcium fosfomycin in the drinking water controlled the adverse effects of experimental colibacillosis., also agreed with (Kowalski, 2007) who reported that thiamphenicol has the potential to become available antibiotic in the treatment and control of a wide range of respiratory and alimentary tract infection of bacterial origin in live stock of mammals, poultry and fish, and (Erener et al., 2010) who reported that Nigella sativa seed was effective against total coliform count in the intestine of broilers.

The mean body weight, Feed conversion rate (FCR) and Average daily gain were significantly improved in chicken treated with fosfomycin, thiamphenicol and Nigella sativa oil when compared with infected non treated group, the best significant performance parameters were demonstrated in groups treated with fosfomicin and thiamphenicol in combination of  Nigella sativa oil  rather than groups treated with each compound separately, our result was similar to findings reported in chicken treated with fosfomycin (Fernandez et al., 1998, 2002), also (Halle et al., 1999; Erener et al., 2010; Shewita and Taha, 2011) showed that dietary black cumin (Nigella sativa) and its oil extract improved feed intake, body weight and FCR in the broilers even at low dose.

Treatment of E. coli O₁₁₁ infected chickens by fosfomycin, thiamphenicol alone or in combination with Nigella sativa oil reduce E.coli reisolation rate from liver, heart and air sac when compared with those data recorded in infected non treated chickens. These findings were supported by previous report (Shaheen and El-Far, 2013) who recorded that combination of the therapeutic doses of pefloxacin and florfenicol reduce E.coli reisolation rate from liver and heart. Also agreed with the result obtained by (Fernandez et al., 1998, 2002) who demonstrated that fosfomycin reduced the numbers of birds from which bacteria were reisolated and it was effective for treatment of E.coli infection in chickens.

The result of antibacterial activity of fosfomycin, thiamphenicol and Nigella sativa oil against E coli O₁₁₁ which measured as diameter of zone of inhibition (mm) showed that the administration of Nigella sativa oil with fosfomycin or thiamphenicol increase their antimicrobial activity. Our result agreed with that obtained by (Hanafy and Hatem, 1991) who recorded that the extract of Nigella sativa showed antibacterial synergism with streptomycin and gentamycin and showed additive antibacterial action with some antibiotics such as spectinomycin, erythromycin, tobramycin and chloramphenicol.

The effect of Nigella Sativa on the immune- response has been investigated by several researches. Many studies have reported that the oil of N.Sativa and its component produce an increase in the T- helper cells and enhance natural killer cell activity, also they had a stimulatory effect on macrophages (El- Kadi and Kandil, 1986 and Haq et al., 1995). The ability of the most abundant component of Nigella Sativa oil; Thymoquinone (TQ) to modulate cytokines and enhance the immune system has been implicated as the main reason for its protective effect against schistosoma egg infection in the liver (Mahmoud      et al., 2002). Dorucu et al. (2009) proved that serum proteins and total immunoglobulin levels were significantly increased in rainbow trout fish.

In this study, the specific antibody titers against the   E. coli O₁₁₁ detected by ELISA in serum were significantly increased after two weeks of infection in 5^th, 6^th and 7^th groups which received the Nigella Sativa oil in comparison to other groups. These results agreed with those of Durrani et al., 2007; Al-Beitawi et al., 2009; Shewita and Taha (2011) and Al-Mufarrej, (2013) who reported significant improvement in antibody titer. Using Nigella Sativa as feed supplementation enhanced antibody production in laying hens (Yalçin et al., 2012). Moreover, using Nigella Sativa oil significantly enhanced the immune system through increased lymphocyte production, and inhibited development of advanced dysplastic changes after topical application of DMBA (7,12- Dimethylbenz(a) anthracene) in hamsters to induce immune-suppression (Al-Jawfi    et al., 2008). The results of this study disagreed with the finding of (Jang, 2011) who recorded no significant effects on immunity parameters except in lymphocyte ratio. In conclusion, the use of Nigella Sativa oil has beneficial effects on the performance, feed intake, body weight and immunological response in the broilers. Future researches should focus on the mechanisms by which the Nigella Sativa oil exerts their effects.

ACKNOWLEDGEMENT

The authors gratefully acknowledge the help and cooperation of Dr. Selim Selim Salama Senior Researcher, Central Laboratory for Evaluation of Veterinary Biologics, Abbasia, Cairo and Dr. Hosni Awaad El-Banna professor of pharmacology, Faculty of Veterinary Medicine, Cairo University for their help and cooperation in this study.

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