Effect of anti-inflammatory drugs on colibacillosis lesions in broilers after Infectious Bronchitis Virus and subsequent Escherichia coli infection

Abstract

Background: In case of persistent and sterile inflammation, anti-inflammatory drugs should be considered as first choice treatment instead of antibiotics.

Objective: To assess the effect of anti-inflammatory drugs on lesions due to colibacillosis.

Animals & methods: Five groups of day-old broilers of 15 birds each were housed in isolators and were inoculated at 29 days of age with Infectious Bronchitis Virus strain M41 by the oculo-nasal and IT (intratracheal) route (10^5.4 EID₅₀ (egg infectious dosis 50)/broiler) and at 33 days of age with Escherichia coli strain 506 by the IT route (10^7.6 colony forming units/broiler). Broilers of four groups were treated from day 28 up to and including day 39 orally on a daily basis with either carbasalate calcium (4 × 12.5 mg), meloxicam (2 × 0.5 mg), acetaminophen (4 × 2.5 mg), or dexamethasone (1 × 1.0 mg). The fifth group was placebo-medicated. At 40 days of age, the experiment was ended and at post-mortem examination, colibacillosis lesions were assessed.

Results: All broilers in the dexamethasone group died. This mortality exceeded significantly (p < 0.05) that of the other groups in which mortality ranged from 2 to 5. Mean lesion score of surviving broilers of medicated groups ranged from 5.3 to 5.8 compared to 3.9 in the placebo group and did not differ significantly between groups.

Conclusion: None of the anti-inflammatory drugs had a positive effect on colibacillosis lesions.

Clinical importance: Anti-inflammatory drugs cannot be considered as an alternative for antibiotic treatment.

Keywords:

• anti-inflammatory drugs
• colibacillosis
• broilers
• chickens
• Infectious Bronchitis Virus
• Escherichia coli

1. Introduction

Colibacillosis caused by avian pathogenic Escherichia coli is one of the major causes of economic and welfare loss in broiler farming worldwide. The disease occurs predominantly from 3–4 weeks of age onwards as superinfection following infection with respiratory agents such as Newcastle Disease Virus and Infectious Bronchitis Virus (IBV) (Goren 1978; Gross 1990; Glisson 1998; Dho-Moulin and Fairbrother 1999).

Outbreaks of colibacillosis in broilers are treated with antibiotics. In the Netherlands, about one third of the antibiotics used in the broiler industry is consumed in these outbreaks (Central Antibiotic Usage Database of GD – Animal Health Service, Deventer, the Netherlands). However, a number of drawbacks is associated with large scale antibiotic use such as residues in the food chain and most important the development of (multi)resistance, which is not only of relevance for the animal species concerned, but also for human health.

Colibacillosis is characterized by fibrinous polyserositis (airsaculitis, pericarditis, perihepatitis, and peritonitis). In superinfected broilers (both IBV and E. coli infected), lesions of the respiratory tract mainly consisting of airsacculitis and pneumonia, are characterized by persistent inflammation, while the clearance of E. coli does not seem to be impaired (Dwars et al. 2009). In case of persistent and sterile inflammation, anti-inflammatory drugs should be considered as first choice treatment instead of antibiotics.

Therefore, in the present study the effect of anti-inflammatory drugs (carbasalate calcium [a mixture of calcium acetylsalicylate and urea], meloxicam, acetaminophen (paracetamol), and dexamethasone) on colibacillosis lesions in broilers following IBV and E. coli inoculation was assessed. Anti-inflammatory effects were determined by colibacillosis lesions scoring, which is a semiquantitative method to macroscopically evaluate the extent of inflammation.

Medication doses and frequency were based on literature (Likoff et al. 1981; Huff et al. 1998, 2004; Baert and De Backer 2003; Jayakumar et al. 2010). In order to maximize the chance of efficacy, drugs were administered long term, i.e. 12 days. A placebo-treated IBV and E. coli inoculated control group was included in the study.

2. Materials and methods

2.1. Experimental chickens

Eighteen-day-incubated eggs originating from a Mycoplasma gallisepticum-free broiler parent flock (Cobb) were obtained from a commercial hatchery and hatched at the research facility of the Faculty of Veterinary Medicine, Utrecht University, the Netherlands.

2.2. Housing, feeding, and management

Broilers were housed in negative pressure HEPA isolators (Beyer and Eggelaar, Utrecht, the Netherlands) each with a volume of 1.38 m³ and fitted with a wire floor of 1.1 m². Isolators were ventilated at a rate of approximately 30 m³/h. Until 14 days of age, broilers were given ad libitum a commercial phase 1 broiler feed containing 11.3 MJ of metabolizable energy and 220 g crude protein per kg. From 15 days of age, a phase 3 feed containing 12.4 MJ of metabolizable energy and 183 g crude protein per kg was provided. The rationale for using a phase 3 feed immediately after a phase 1 feed was to avoid stress due to a change in feed type during the actual experiment. In order to prevent leg disorders and hydrops ascites the feed was given on “skip a day” basis, whereby during the feeding day 150% of the ad libitum daily intake obtained from the breeder's manual was given. This percentage was increased to 170% from 23 days of age onwards to compensate for the relative low protein content of the phase 3 feed, which is normally given after approximately 35 days of age.

Tap water was provided ad libitum throughout the experimental period. Until 15 days of age 22-h light was given per 24 h. Thereafter it was reduced to 16 h. At the age of 4 days onwards the color of lighting was changed to red to prevent cannibalism. Isolator temperature was gradually decreased from 35°C at day 1 to 20°C at day 31 after which the isolator temperature was kept at 20°C.

2.3. Inocula

The virulent Massachusetts IBV strain M41 was obtained from Intervet B.V., Boxmeer, the Netherlands, as freeze-dried allantoic fluid in vials containing 10^7.3 EID₅₀ (egg infectious dosis 50)/0.2 mL. The virus had undergone seven passages in SPF-embryonated eggs. All IBV inocula were prepared just prior to use in distilled water to a concentration of 10^5.3 EID₅₀/mL.

The E. coli strain 506 (O78; K80) is a flumequine resistant strain isolated from an inflamed pericardium of a commercial broiler suffering from natural colibacillosis (Van Eck and Goren 1991). The ability of the E. coli 506 strain to induce colibacillosis in SPF chickens and commercial broilers, which had been exposed previously to live IB or Newcastle disease vaccine virus, was demonstrated earlier (Goren 1978; Van Eck and Goren 1991). The E. coli culture was prepared as described by Matthijs et al. (2003) and was diluted 1 : 10 in phosphate-buffered saline, resulting in a concentration of 10^7.6 colony forming units (CFU)/mL.

2.4. Experimental design, inoculations, and treatments

The experimental design has been outlined in Table 1. At day 1, broilers were randomly divided into five groups of 15 chickens each, which were housed in separate isolators. At day 29, broilers of all groups were IBV inoculated by the oculo-nasal (0.2 mL/bird) and intratracheal (IT) (1.0 mL/bird) route resulting in a dose of 10^5.4 EID₅₀/bird. The E. coli 506 strain was administered to all groups IT at day 33 in a dose of 10^7.6 CFU/bird in a volume of 1.0 mL. IT application of IBV and E. coli was performed using a 1.0 mL syringe fitted with a blunt-ended pipette tip (Corning, NY, cat. No. 4862, USA).

Table 1. Effect of orally administered anti-inflammatory drugs on colibacillosis lesion score in commercial broilers inoculated with IBV M41 and E. coli 506 at 29 and 33 days of age, respectively. The experiment was ended at 40 days of age.

	Treatment^B,C					Surviving chicks at 40 days of age
Group^A	Compound	Bolus dose (mg/broiler)	No. Boli per day	Duration (days)	Mortality^D*	MLS^E*	Mean HI IBV titer ± SD (log2)^F*
1	Carbasalate calcium	12.5	4	12	5^a	5.8^a	10.4 ± 0.9^a
2	Meloxicam	0.5	2	12	1^a	5.6^a	10.5 ± 1.1^a
3	Acetaminophen	2.5	4	12	2^a	5.3^a	10.1 ± 1.2^a
4	Dexamethasone	1.0	1	12	15^b	–	–
5	Distilled water (placebo)	–	2	12	2^a	3.9^a	10.1 ± 1.4^a
Notes: ^AEach group consisted of 15 birds.
^BAll treatments started at 28 days of age; all boli had a volume of 1.0 mL.
^COverall mean body weight ± SD was 718 ± 68 g at 28 days of age and 1285 ± 170 g at 40 days of age, without significant differences between groups.
^DExcept for two broilers in group 4, all mortality occurred after E. coli inoculation and was specific (Section 2.9).
^EMean lesion score.
^FHI antibodies against IBV were not detected at 29 days of age in any of the groups (HI titers ≤4 (log2)).
*Values with dissimilar superscripts differ significantly (p < 0.05).

All treatments started one day before IBV inoculations (day 28). Groups of broilers were orally given carbasalate calcium (Ascal 600, Meda Pharma B.V., Amstelveen, the Netherlands) (Group 1), meloxicam (Metacam suspension 15 mg/mL, batchnr. 059ZV20, Boeringer Ingelheim B.V., Alkmaar, the Netherlands) (group 2), acetaminophen (Daro Paracetamol sirope 120 mg/5 mL, Remark Pharma, Meppel, the Netherlands) (group 3), or dexamethasone (Rapidexon 2 mg/mL, batchnr. W307, Eurovet Animal Health B.V., Bladel, the Netherlands) (group 4). All drugs were prepared using distilled water with the bolus dose in a volume of 1.0 mL and administered in the oesophagus using a 2 mL syringe coupled to a knobbed-curved stainless steel cannula (art. nr. 14186, AUV Group, Cuijk, the Netherlands). Boli were prepared freshly before each application.

Drugs were administered one, two, or four times a day at 12.30 h; at 8.00 and 21.30 h; and at 8.00, 12.30, 17.00, and 21.30 h, respectively. The bolus dose (mg/broiler) and frequency of daily treatments can be found in Table 1. All treatments lasted 12 days, i.e. up to and including the day before ending the experiment (Table 1).

A placebo-treated control group (group 5), which was given 1.0 mL distilled water orally twice daily, was included in the experiment. At 40 days of age the experiment was ended. Surviving broilers were stunned using electric current (10 s at 220 V) and debleeded by incision of the vena jugularis. Both, surviving birds and those that died during the experiment were subjected to post-mortem examination.

2.5. Clinical observations

Clinical inspection and removal of dead birds was performed daily. Dead birds were stored at −20°C until post-mortem examination at day 40. At day 33 (4 days after IBV inoculation), all birds were individually examined for the presence of mucous nasal discharge, a prominent clinical sign of IBV infection. Hereto mild pressure was applied directed from the nasal bone region toward the nostrils (Matthijs et al. 2003). A bird was recorded as having nasal discharge when this clinical sign was observed on either one or both sides.

2.6. Bodyweight

During the experiment a fixed dose for each treatment was given. In order to calculate the corresponding dose per kg body weight, broilers were weighed at 28 and 40 days of age.

2.7. Serological examination

At day 29 (day of IBV inoculation) and day 40 (end of the experiment), blood samples were collected from all broilers by puncturing the ulnar vein at day 29 and the jugular vein at day 40. Blood samples were examined for hemagglutination inhibition (HI) antibodies to IBV M41. The HI test using eight hemagglutinating units was performed as described by Alexander and Chettle (1977). Titers are expressed as log₂ of the reciprocal value of the highest serum dilution showing complete HI. Titers exceeding log₂ 4 were considered positive.

2.8. Post-mortem examination

At day 40, birds were euthanized and post-mortem examination was performed. Special attention was paid to colibacillosis lesions, which were scored macroscopically in the following organs: right thoracic air sac, left thoracic air sac, pericardium, and liver.

Lesion scoring was performed as described by Van Eck and Goren (1991) using the following criteria: 0 = no lesions; 0.5 = one yellow/brown pinhead-sized spot indicative of inflammation; 1 = two or more pinhead-sized spots as described at score 0.5; 2 = thin layer of fibrinous exudation on various locations; and 3 = thick and extensive layer of fibrinous exudation. The maximum score per bird is 12. Birds with lesions in pericardium and/or liver were considered as generalized infected. Mean lesion scores (MLS) were calculated per group. Examinations were performed blindly. Birds that died during the experiment were also necropsied, but their lesion scores were not included in the calculation of MLS.

2.9. Bacteriological analysis

After post-mortem examination, femurs of birds that had died after the E. coli inoculation were collected and bacteriological analysis of the bone marrow was performed. Culturing was done using sheep blood agar plates and colonies were identified using Bactident® E. coli (no. 13303; Merck, Amsterdam, the Netherlands), as described by Matthijs et al. (2003). Mortality was considered specific, i.e. due to the E. coli infection, in case birds showed generalized colibacillosis and/or bacteriological analysis of bone marrow was positive.

2.10. Statistical analysis

Differences in mean body weight between groups were assessed using the one-way ANOVA test and Bonferroni as post hoc test, while the mean colibacillosis lesion scores and HI IBV M41 antibody titers were analysed using the non-parametric Kruskal–Wallis test. Differences in mortality between groups were analysed using Fisher's exact test. Differences were considered significant when p < 0.05 (Statistix 2010).

2.11. Ethics

Birds were housed, handled, and treated following approval by the Animal Experimental Committee of Utrecht University, the Netherlands, in accordance with the Dutch regulation on experimental animals.

3. Results

3.1. Clinical disease signs

Four days after IBV inoculation almost all birds showed mucous nasal discharge. Birds were slightly to moderately depressed from 2 days after E. coli inoculation until the end of the experiment.

3.2. Body weight and treatment dose per kg body weight

Mean body weight ± SD was 718 ± 68 g at 28 days of age and 1285 ± 170 g at 40 days of age, without significant differences between groups. Due to increasing body weights with time the applied treatment dose per kg body weight decreased accordingly. The bolus dose in mg per kg body weight decreased from 17.4 to 9.7 for carbasalate calcium, from 0.7 to 0.4 for meloxicam, from 3.5 to 1.9 for acetaminophen, and from 1.4 to 0.8 for dexamethasone, at 28 and 40 days of age, respectively.

3.3. Serology

Antibodies to IBV were not detected in any of the groups at 29 days of age (HI IBV log₂ titers ≤4). At day 40, mean HI IBV log₂ titer ± SD varied from 10.1 ± 1.4 to 10.5 ± 1.1 between groups. Differences between groups were not statistically different (Table 1).

3.4. Mortality and post-mortem examination

Two broilers of the dexamethasone group died at day 31, i.e. before the E. coli inoculation, due to fatty liver degeneration and dilation of the right heart, respectively. All other mortality occurred 2–7 days after E. coli inoculation, was specific, and varied from 2 to 5 between groups 1, 2, 3, and 5. Differences between these groups were not statistically significant. In group 4 (dexamethasone group) all birds died. The MLS of broilers that died in this group after E. coli inoculation (n = 13) was 4.9. Mortality in group 4 exceeded significantly (p < 0.05) that of all other groups.

MLS of surviving chickens in the placebo-medicated control group (group 5) was 3.9, while the MLS of the other groups ranged from 5.3 to 5.8. Differences between groups were not significant (p > 0.05) (Table 1).

4. Discussion

In the placebo-treated IBV and E. coli-inoculated control broilers, the MLS was 3.9, which was in agreement with previously performed experiments (range 3.1–4.7) (Matthijs et al. 2003; 2005) reflecting the good reproducibility of the model used.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as salicylates, acetaminophen, and meloxicam act as anti-inflammatory, antipyretic, and analgesic agents by inhibition of prostaglandin synthesis (Vane and Botting 1995; Graham and Scott 2005). Beneficial effects on mortality and airsac lesions following experimentally induced colibacillosis were observed in broilers and turkeys by treatment with aspirine (acetylsalicylic acid) and sodium salicylate in doses equal to the dose used in the present study (Likoff et al. 1981; Huff et al. 2004). However, unexpectedly, in our study none of the NSAIDs showed a positive effect on colibacillosis lesions. This discrepancy may have been the consequence of a number of differences in the experimental design. For example, the lack of IBV in the infection model, the use of other routes for the inoculation of E. coli, other doses of E. coli, distinct administration routes of drugs and in one study the use of turkeys instead of chickens. Studies on the effect of acetaminophen and meloxicam on colibacillosis in poultry have not been published to date. Although not significantly (p > 0.05), all NSAIDs used in the present study increased the MLS of colibacillosis. Exacerbation of inflammatory conditions in poultry due to medication with NSAIDs have not been described yet, but this finding has been reported in both laboratory mammals (Reuter et al. 1996; Tanaka et al. 2009) and in man (Kefalakes et al. 2009).

Dexamethasone is a synthetic glucocorticosteroid with well-know potent anti-inflammatory and immunosuppressive properties (Huff et al. 1998; Saklatvala 2002; Aengwanich 2007). The immunosuppressive capabilities provide a good explanation for the 100% mortality found, which was largely caused by bacteremia. However, a reduction in inflammatory lesions did not occur as the MLS in these birds was even higher than that of control broilers, although not statistically significant.

In conclusion, none of the anti-inflammatory drugs had a positive effect on colibacillosis lesions and consequently, they lack practical relevance for the field and cannot be considered as an alternative for antibiotic treatment. Even in case treatment with these drugs used at a different time point during disease (i.e. only during the early phase of IBV infection or only during persistent E. coli inflammation) would yield promising results, their applicability under field circumstances would remain questionable as in a flock birds with various stages of the disease always occur.

Acknowledgment

We thank Vincent Polak for his assistance with the animal experiment.