In vitro and in vivo evaluation of antimicrobials in Escherichia coli infection in broilers and evaluation of ciprofloxacin in induced colibacillosis

The present study was conducted to 1) Survey and record the data regarding use of antimicrobials in Escherichia coli infection in broiler poultry flocks 2) Evaluate the sensitivity of E. coli against antibiotics and 3) Evaluate the ciprofloxacin in induced colibacillosis. For this purpose, fifty (50) broiler poultry farms were surveyed and in vitro evaluation of antimicrobials in E. coli infection was evaluated. The farmer level data regarding awareness about antibiotic usage pattern, storage of antibiotics, withdrawal period and antibiotic resistance was evaluated. Data revealed that most of broiler poultry farmers relied on quinolones (especially ciprofloxacin) to combat E. coli infection. The morbid samples from the E. coli infected farms were collected, pathogenicity of E. coli was checked and sensitivity against seven antibiotics viz. ciprofloxacin, enrofloxacin, gentamicin, oxytetracycline, colistin, neomycin and amoxycillin was evaluated. Sensitivity tests showed E. coli was highly sensitive against ciprofloxacin, enrofloxacin, and gentamicin. On the basis of antibiogram, the efficacy of ciprofloxacin was determined under in vivo conditions. Accordingly, 150 Hubbard (un-sexed) broiler chicks were divided into three groups viz. A, B & C each having 50 chicks; group A was kept as uninfected and untreated (control), group B was experimentally infected and treated with ciprofloxacin while group C was infected and kept untreated. The mortality in group A, B and C was recorded as 2, 14 & 28 %, respectively. The cumulative feed consumption in group A, B and C was 84, 87 & 86 kg respectively. Mean weight gain in group A, B and C was 882.47, 868.70 & 801.29 gm, respectively. The FCR in group A, B and C was 1.9, 2 and 2.15, respectively. It was concluded that the use of ciprofloxacin had high efficacy against E. coli infection in broiler chicks.


Introduction
Poultry farming is one of the most profitable businesses in Pakistan.Poultry sector is the second largest industry in Pakistan after textile.It has emerged as a cheap and economical substitute for beef and mutton in Pakistan.Its importance can be judged from the fact that almost every family in rural areas and every fifth family in urban areas is associated with poultry production activities in one way or the other.The poultry industry has developed rapidly over the last three decades with an impressive growth rate of over 8% [1].The development, expansion and intensification of poultry has faced problems of disease, nutrition and management.Colibacillosis caused by E. coli is a disease of economic concern in poultry industry [2-4].E. coli is responsible for a variety of poultry diseases as it comprises of non-pathogenic and pathogenic strains that cause a number of diseases in host species including colibacillosis, colisepticemia, Hijar's disease, coligranuloma, peritonitis, salpingitis, synovitis, omphalitis and air sac disease [5].E. coli is also responsible for high chick mortality, air saculitis, pericarditis and may predispose bird to adenovirus, reovirus, coronavirus and IBD virus and other secondary bacterial infection [6].An incidence of 52% of colibacillosis is reported through isolation and identification of E. coli from trachea, lung, heart, blood, liver and spleen of poultry birds [7].The prevalence of colibacillosis of 1% and 0.5% in 25-30 days old and 31-35 days old broilers, respectively is observed [8].Antimicrobial therapy is an important tool in reducing the losses due to incidence and mortality associated with avian colibacillosis [9,10,11].The fluoroquinolones are a class of antimicrobials that exhibit excellent activity against gramnegative organisms [12].Among different fluoro-quinolones ciprofloxacin is of the potential interest for veterinary medicine.It has a wide antibacterial spectrum against mycoplasma, salmonella, E. coli, Haemophilus paragalinarum, Pasteurella multocida and staphylococcal infections in chicks [13,14].Quinolones are bactericidal and act by inhibiting bacterial DNA replication and transcription.To facilitate coiling, winding and unwinding, the DNA gyrase allows the stands to be cut and reconnect.DNA gyrase, a topoisomerase, consists of A and B subunits.The most common target site for quinolones is the Asubunit of DNA gyrase coded by the gene gyrA.In Pakistan, the efficacy of this drug has not been evaluated.The present study was designed to determine in vitro and in vivo efficacy of ciprofloxacin in broiler chicks against E. coli.

Survey for E. coli infected farms
Fifty (50) broiler farms located in district Toba Tek Singh were visited for collection of data.Toba Tek Singh is located between 30°33' to 31°2' degree north latitudes and 72°08' to 72°48' degree longitudes.Data regarding drinking water, feeding, vaccination, disease-related morbidity, mortality and antibiotic usage, duration, efficacy, storage, brand, price and with drawl period were collected with an emphasis on E. coli infection.The efficacy of ciprofloxacin medication was determined.

Isolation of E. coli
The livers and hearts of sick/dead chicks suffering from E. coli infection were collected.Samples were transported to the laboratory and processed as soon as possible for the isolation of E. coli.The MacConkey's agar medium, triple sugar iron agar medium and Simon's citrate medium were used for isolation, purification and identification of the organism [15].The morphological, cultural staining characteristics, sugar fermentation, biochemical properties of isolates were determined.Biochemical tests were conducted according to recommended procedures [15][16][17].

Pathogenicity of E. coli
A gut loop assay was performed for the determination of pathogenicity [18].The rabbits were anaesthetized with ketamine HCl and ileum was exposed.½ mL culture of the test isolate was delivered into the lumen of the intestine.An equal volume of normal saline was injected into the intestine of control rabbits.The cavity was sutured with catgut and animals were kept under extreme care and observations were recorded.

Antibiotic sensitivity of E. coli
The susceptibility of pathogenic isolates to seven different antibiotics was determined using the disc diffusion method [19].Mueller-Hinton agar was used for antibiotic susceptibility testing.Nutrient agar was incubated at 37 o C for 15 hrs.The growth was diluted with normal saline to a density visually equal to 0.5 Mc-Farland.Using a sterile swab, the bacterial suspension was spread in 3 different plates on Mueller-Hinton agar.The plates were dried at room temperature for 5 minutes.Seven commercially available antibiotic discs i.e. amoxicillin, gentamicin, ciprofloxacin, oxytetracycline, colistin, enrofloxacin and neomycin were placed on the agar plates using sterile forceps.The plates were incubated at 37 o C for overnight.The radius of zone of inhibition of each antibiotic was measured using Schering antibiotic zone gauge and interpreted according to the zone size interpretative chart.Staphylococcus aureus ATCC 25923 (American Type Culture Collection) was used as a quality control organism.The resistant, intermediately susceptible and fully susceptible isolates to each antibiotic were measured and recorded (Table 1).

Evaluation of ciprofloxacin in induced E. coli infection
For evaluation of ciprofloxacin in induced E. coli infection one hundred and fifty (150) day-old Hubbard (un-sexed) broiler chicks were reared under uniform conditions of management and nutrition at department of Veterinary Clinical Medicine and Surgery, University of Agriculture, Faisalabad.All chicks were vaccinated against Newcastle disease, Infectious bursal disease (IBD / Gumboro) and Hydropericardium syndrome (Table 2).At 20 th day of age, chicks were randomly divided into three equal groups viz.A, B and C. 1mL of diluted broth culture of a pathogenic strain of E. coli (1 × 10 6 CFU organisms / mL) was inoculated orally with the help of the crop tube to the chicks of group B and C, while the chicks of group A were kept as control (uninfected & untreated).All the chicks were kept under observation for development of clinical signs and symptoms for 2 weeks.After the appearance of clinical signs (dullness, depression, vent pasting, respiratory and enteric signs) and postmortem lesions (pericarditis, perihepatitis and air sacculitis), the chicks of group B were treated with ciprofloxacin 20% @ 1 ml / 4L (Ciprosel ® ) PO as recommended by the manufacturer for 5 days.Chicks in group C were challenged and untreated control, while group A was control (unchallenged and untreated) (Table 3).

Pathogenicity of E. coli
In nutrient broth E. coli isolates produced slight pellicle formation at 18-24 hrs with a slimy deposit at the bottom of the tube which on shaking produced a uniform turbidity.The identified culture of E. coli was tested by gut loop ligation method for pathogenicity of the strain.In the positive cases intestinal part became triple size as compared to the normal.The fluid accumulation in intestine indicated positive results, whereas, absence of fluid accumulation indicated negative results.
Along with the presence of fluid, the amount of the fluid also served as an indicator of positive results.The higher the amount of fluid observed, the higher the pathogenic strain of E. coli it represents.The culture that showed maximum pathogenicity was selected for induced E. coli infection.The control sample was inoculated with normal saline instead of E. coli.In the present study intestinal loop ligation method positive (i.e., pathogenic) E. coli were identified by increased size (i.e triple fold than normal) and increased luminal fluid of the intestine, which coincide with the results of previous studies [18 , 23, 26].

In vivo efficacy of ciprofloxacin in induced E. coli infection
In our study the artificial infection of colibacillosis was done using pathogenic isolate of E. coli which was inoculated @ 1 × 10

Mortality post-treatment
The mortality in group A, B and C was 1, 7 and 14 chicks, respectively after treatment (Table 7).

Postmortem findings
The post-mortem examination of chicks died in different experimental groups was performed.Thickened and cloudy air sacs were observed.In severe cases, caseous exudates on air sacs were present.Adhesive pericarditis and fibrinous perihepatitis were present in most of the cases.Enteritis along with excessive mucus was common.The primary lesions of colisepticemia were pericarditis, perihepatitis and air sacculitis were observed.Pericardial fluid became progressively more fibrinous.Necrotic foci in the heart muscles were present in chicks that succumbed to infection.The pericardial sac was thickened with pale-colored gelatinous exudates.Congestion of liver, spleen, kidney and small intestine were also observed.Similar findings were also observed in previous studies [14,23].
The cumulative feed consumption, per bird feed consumption, mean body weight of chick sand FCR in each group is presented in (Table 8).FCR of broiler chicks kept in the group A, B, and C were 1.90, 2.00 and 2.15 respectively.FCR increased in group C because chicks in this group were infected with E. coli but were not treated.

Table 1 . Disc potencies of antibiotics, inhibition zone radius (mm) to categorize E. coli and inhibition zone size of quality control organism (S. aureus ATTC 25923) Name of Antibiotic Symbol Disc Poten cy (µg / disc) Inhibition zone radius (mm) to categorize E. coli & interpretation Inhibition zone size (mm) of quality control organism (S. aureus ATTC 25923)
[15]for overnight.The viable bacteria were counted[15].The broth was diluted with phosphate buffer saline (PBS) to have approximately 1 × 10 6 CFU organisms / mL, which was used for experimental infection.

Table 2 . Vaccination schedule of experimental chicks
E/D Eye drop; S/C Sub-cutaneous; D/W Drinking water

Table 3 . Different treatments in experimental groups
Survey report regarding treatment of E. coli infection in broiler birds is shown in (Table4).Frequency distribution (range of poultry farmers) of awareness about antimicrobial therapy is shown in (Table5).