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Overview of Research with Aureomycin Plus BMD for Ileitis

by 5m Editor
6 January 2009, at 12:00am

Pig veterinary consultant, Dr Nate Winkelman and Dr Teddi Wolf of Alpharma review the research into the combination of Aureomycin® and BMD® to treat ileitis. The paper was presented at Alpharma's Swine Enteric Health Symposium 2008.

Introduction

Porcine proliferative enteropathy (PPE), also known as ileitis, is an enteric disease of growing and finishing pigs worldwide. It is considered to be ubiquitous in US grower-finisher operations.

The disease is characterized by a thickening of the mucosa of primarily the small intestine. Patho-gnomonic lesions consist of proliferative immature crypt cells in the intestinal epithelium and the presence of free floating bacteria in the apical cytoplasm. The causative agent is the obligate intracellular gram-negative bacterium, Lawsonia intracellularis. Clinical ileitis is manifested by inappetance, diarrhoea, poor performance and mortality, and sub-clinical ileitis can also occur. Both forms of ileitis can be costly, with estimates ranging from $4.00 per pig for sub-clinical disease to $22.00 per pig in clinical outbreaks.9,17 With feed prices at an all-time high, these estimates are likely conservative.

Non-vaccinational control of ileitis consists largely of feedgrade antimicrobial applications. There are five feed-grade agents approved for treatment or control of ileitis.

The Alpharma control program involves feeding Aureomycin chlortetracycline (CTC) at 10 mg/lb bodyweight (BW) plus BMD at 30 grams/ton (see Aureomycin 10 mg/lb BW calculation table at the end of this article). The combination is approved for control of porcine proliferative enteropathies (ileitis) caused by Lawsonia intracellularis susceptible to chlortetracycline (feed for 14 days). The combination is also approved for treatment of bacterial enteritis caused by Escherichia coli and Salmonella choleraesuis and bacterial pneumonia caused by Pasteurella multocida organisms susceptible to chlortetracycline and for increased rate of weight gain and improved feed efficiency.3

In-Vitro and In-Vivo Research

The tetracyclines are usually associated with respiratory disease treatment or control. Indeed, CTC has a broad spectrum of efficacy against susceptible strains of many swine respiratory disease pathogens, including Mycoplasma hyopneumoniae, Pasteurella multocida, Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, and Bordetella bronchiseptica (Table 1).7, 12-15

In addition, CTC’s pharmacokinetic / pharmacodynamic characteristics, resulting in high blood and lung tissue levels, support its use for respiratory disease control. CTC’s enteric disease efficacy, and in particular, efficacy against Lawsonia intracellularis, is less well recognized and is the focus of this paper.

CTC is well absorbed after oral administration and concentrates in cell cytoplasm where it works to inhibit bacterial protein synthesis. CTC is effective against other intracellular organisms such as Anaplasma marginale in cattle, Chlamydia spp. in swine, and rickettsiae in dogs. Historically, the MIC of CTC for Lawsonia is low at <1 μg/mL.11,12

Pre-dating live animal research efforts, there were anecdotal reports of ileitis control with CTC from swine practitioners in the field.4 In the mid-90’s, live animal research was conducted using a hamster challenge model to demonstrate that Aureomycin could control the clinical signs and lesions associ ated with Lawsonia infection.5

Several studies in swine soon followed. McOrist used his pure culture challenge model to show that Aureomycin fed to pigs at 300 or 600 ppm (approximately 8 and 18 mg/lb BW) was more effective than Tylan® (Elanco Animal Health) at 100 ppm in preventing clinical ileitis and providing significant improvements in average daily gain versus challenged, non-medicated controls.10

Winkelman, using his mucosal homogenate challenge model, fed Aureomycin at low (100 grams/ton) or therapeutic (10 mg/lb BW) levels, starting prior to, or at the onset of, clinical signs. Both dosage levels and dosing regimens were effective but due to small numbers of pigs, it was impossible to differentiate between dosage levels.22

Winkelman went on to evaluate BMD plus CTC (in this case, Alpharma’s ChlorMax®) in his mucosal homogenate challenge model. BMD was fed at 30 grams/ton and CTC at 10 mg/lb BW. This study showed the effectiveness of CTC in reducing the clinical signs and lesions of ileitis, and also demonstrated synergy between BMD and CTC. The incidence of severe ileal/ jejunal lesions was reduced (P < 0.05) by 14 per cent in the BMD plus CTC treatment group vs. pigs fed only CTC.23 Next, Winkelman compared the effectiveness of ileitis control programs utilizing BMD plus Aureomycin, Tylan or Lincomix® (Pfizer Animal Health).24 This study is described in more detail later in this paper.

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More About Bacitracin and CTC Synergy

Researchers have helped to shed some light on why the synergistic combination of bacitracin and CTC often works better than bacitracin or CTC alone. In 1975, Walton and Bird published groundbreaking research that explained the mechanism of growth promotion caused by bacitracin. They studied the effect of bacitracin on the cell wall of E. coli and discovered that the antibiotic caused herniation of cell contents at one end of the bacterium and areas of cell wall damage along the long axis of the cell. Walton postulated that these defects would predispose the cell to lysis and make the bacterium more susceptible to the host’s immune system. He concluded that this may be part of the growth promotion effect of bacitracin. Not only were gram-positive bacterial flora altered to improve feed utilization, but gram-negative bacterial numbers were reduced at sites of high bacitracin concentration in the gut, resulting in increased nutrient availability as well.20

Walton later studied the impact of bacitracin on the MIC of various antibiotics on several human and animal bacterial isolates, including E. coli and Salmonella typhimurium. The sensitivity of S. typhimurium to tetracycline was increased in the presence of bacitracin. Walton concluded that since bacterial sensitivity to tetracycline can be increased by higher permeability of the cell wall, the increased sensitivity of S. typhimurium may be due to damage to the cell wall caused by bacitracin, resulting in increased permeability to tetracycline. 18,19, 21

In vitro work by Mathers and Benning confirmed that there is a synergistic effect for bacitracin and CTC. In a survey of 51 field isolates of Clostridium perfringens, they demonstrated that for selected strains with resistant MIC values for bacitracin and CTC, MIC values were reduced to sensitive levels for the combination.8

More recently, Gebhart investigated the synergistic effects of bacitracin and CTC on Lawsonia intracellularis. The MIC of bacitracin for this isolate was >128 μg/mL and the MIC for CTC was 8 μg/mL. When the isolate was exposed to both bacitracin and CTC, Gebhart found that the MIC of CTC for L. intracellularis was reduced to 2 μg/mL.6

Evaluation of Three Ileitis Control Programs

Study Objective

This study was designed to evaluate the efficacy of BMD plus Aureomycin, Tylan or Lincomix for the control of ileitis. All treatments were fed according to label, starting 3 days prior to challenge (Table 2). Pigs were evaluated for average daily gain and average daily feed consumption. Other parameters such as abdominal appearance, pig attitude, severity of diarrhoea, PPE intestinal lesion scores, PPE lesion length, and mortality were recorded. Immunohistochemistry (IHC) was conducted on ileal samples, and serology and fecal PCR’s were performed. The duration of the study was 25 days.


*All pigs were challenged on day 0 and necropsied on days 21/22

Test Materials

The basal feed was formulated for an 18.5 per cent crude protein (CP) diet. BMD 30 (Alpharma Animal Health), containing bacitracin methylene disalicylate at 30 grams/pound, was added to the diet at the rate of 1 lb per ton of complete feed to provide a 30 gram/ton level of BMD. Aureomycin 90 Granular Premix, containing chlortetracycline at 90 grams/lb, was added to the diet at the rate of 5.6 lb per ton of complete feed to provide 10 mg/lb BW CTC (500 grams/ton). Tylan was added to the feed to provide 100 or 40 grams tylosin phosphate per ton of complete feed. Lincomix was added to the feed to provide 100 or 40 grams lincomycin per ton of complete feed. The feed was assayed prior to study initiation to confirm that drug levels met specifications. The feed was bagged and bag labels colour-coded by treatment so that the investigators were blind to the treatments.

Animals

One hundred sixty healthy commercial crossbred pigs of mixed gender, approximately 5 weeks of age, were brought to the trial site, weighed and ear-tagged. The average pig weight was 26.8 lb (range 22.2-31.4 lb). The pigs were purchased from a “high health” Isowean system free of apparent enteric diseases, including PPE, Brachyspira pilosicoli, Brachyspira hyodysenteriae, TGE, and Salmonella choleraesuis. Likewise the pigs were free of respiratory diseases such as PRRSV (porcine reproductive and respiratory syndrome virus), SIV (swine influenza virus), Actinobacillus pleuropneumoniae, and Mycoplasma. Pigs were acclimated for 4 days, then challenged with L. intracellularis organisms.

Study Facilities

The research site was located near Morris, Minnesota. The facilities were comprised of two identical Double L nursery buildings leased to Swine Services Unlimited Inc. The site was isolated and had no other pigs on it. Each building contained 16 pens measuring 6 feet x 5.5 feet. Footbaths with ammonium chloride disinfectants were used. Pens in the facility were divided by solid plastic partitions, and the pen floors were perforated plastic Poly-grate. One individual five-hole feeder and two nipple waterers were provided in each pen. Pig density at 5 pigs per pen provided 6.6 square feet per pig. The ventilation system was negative pressure, mechanically ventilated with integrated heater and fan controllers. To prevent cross-contamination between groups, separate disposable plastic boots and gloves were used in each pen when clinical scores, faecal and blood samples were obtained.

Study Design

The 160 pigs were blocked by weight and then randomly allotted to one of four treatment groups, with eight pens (replicates) per treatment and five pigs per pen.

Treatments included: 1) non-medicated control; 2) Tylan 100 grams/ton fed for 21 days (D3 through D17), followed by Tylan 40 grams/ton for the remaining 4 or 5 days; 3) Lincomix 100 grams/ton fed for 21 days (D3 through D17), followed by Lincomix 40 grams/ton for the remaining 4 or 5 days; and 4) BMD 30 grams/ton plus Aureomycin 10 mg/lb BW (500 grams/ton) fed for 14 days, starting D-3 and continuing through D10, followed by BMD 30 grams/ton for 11 or 12 days (D11 through D21 or D22) (Table 2).

All pigs were individually weighed on D-3, 10, 17, and 21 or 22, and feed weighback was conducted at the same times. Two pigs per pen were randomly selected before the study started and blood and fecal samples collected from these pre-selected pigs at the start and end of the study. All pigs were challenged orally via feeding tube on day 0. All pigs were evaluated clinically 3 times per week by the investigator. Due to the large number of pigs involved in this study, half of the surviving pigs were humanely euthanized and necropsied on D21 and the remainder on D22.

Mucosal Homogenate Challenge

The L. intracellularis challenge inoculum was prepared according to a standard operation procedure. After preparation, a sample of the mucosal homogenate material was submitted to the University of Minnesota Diagnostic Laboratory for the isolation and enumeration of L. intracellularis, and to confirm the absence of Brachyspira hyodysenteriae, B. pilosicoli, Salmonella choleraesuis and B-hemolytic E. coli. On D0, 50 mL of the mucosal homogenate challenge material containing approximately 4.4x108 L. intracellularis organisms was administered into the esophagus of each pig via a #14 French 41 cm Sovereign feeding tube affixed to a large syringe.

Parameters Measured

  1. Mortality: Pigs that died were necropsied and cause of death determined. Pigs that became moribund during the study were humanely euthanized according to University of Minnesota Animal Usage Form guidelines, necropsied and cause of death determined.

  2. ADG, ADFI, and F/G: Pigs were individually weighed at the start of the trial (D-3), then again on D7, D14, and on day of necropsy (D21 or D22). Weighed amounts of feed were added to each pen daily. Feed remaining was weighed back weekly to permit calculation of weekly pen feed consumption and average daily feed intake.

  3. Clinical evaluations: Pigs were clinically evaluated three times per week by the investigator who was blind to the treatments, and each pig assigned a ‘pig attitude score’, with 1=normal, 2=slight to moderately depressed, listless, and 3=severely depressed, recumbent; an ‘abdominal appearance score’, with 1=normal; 2=moderately gaunt; 3=severely gaunt; and ‘diarrhoea score’, with 1= normal, no diarrhoea, 2=semi-loose, normal color, no blood, 3=watery, voluminous, no blood, 4=blood-tinged, loose or formed, 5=profuse diarrhoea with blood or dark, tarry faeces.

  4. Lesion scores and lesion length: Gross pathology scores were assigned separately to the jejunum, ileum, caecum and colon for all pigs which were euthanized at the end of the study or died during the study. Lesions were scored using a 0-3 grading system, with 0=normal, 1=mild oedema and hyperaemia, 2=oedema, hyperaemia, reticulated serosa and mucosa (PIA lesion), 3=oedema, hyperaemia, reticulated serosa and mucosa, and gross thickening of the mucosa (necrotic enteritis lesion). Gross lesion length was measured in centimetres and recorded for the jejunum, ileum, caecum and colon for all pigs that were euthanized or died.

  5. Diagnostic evaluations: Blood and faecal samples were collected from two pre-selected pigs per pen at the beginning and end of the study for IPMA and PCR evaluation, respectively. At necropsy, 2 to 4 inches of terminal ileum including the ileo-caecalcolonic junction from each pig was saved and fixed in buffered formalin for histologic and IHC evaluation by the University of Minnesota Veterinary Diagnostic Laboratory. The IHC of tissue is the most sensitive method for detection of Lawsonia. The following scoring system was used for IHC evaluation: 0=no specific staining detected, 1+= isolated focus of staining found, 2+=multifocal staining found, 3+ =more than 40 per cent of the glands stained, 4+=all of the glands stained.

Statistical Analysis

The study was conducted using a completely randomized design. A one-way treatment structure was utilized. The model contained only the fixed effect of treatment. The data were analyzed using the General Linear Model (GLM) procedure of SAS (SAS Institute Inc., Cary, NC). When treatment effects were considered significant (P<0.1 or P<0.05), means were separated using t-tests, equivalent to Fisher’s least-significant-difference test (LSD). The statistical analysis was conducted with the statistician blinded to the treatments.

Study Results

All three medications prevented any PPE-related mortality, while 15 per cent of the nonmedicated controls died from PPE (Figure 1). All the medicated treatments showed significant or numerical improvements in performance (Figures 2-4), and the BMD plus Aureomycin pigs showed significant improvements in ADG and ADFI compared to the Lincomix and/or Tylan groups.

All three medications significantly reduced the signs of clinical ileitis compared to the nonmedicated control pigs, and the BMD plus Aureomycin treatment gave significantly better reductions versus Tylan relative to the abdominal appearance score and diarrhoea score (Figure 5). All three medications gave significant or numerical reductions in the incidence and severity of ileal/jejunal lesions compared to the non-medicated controls, as evidenced by lower ileal/jejunal scores, reduced lesion length, and decreased incidence of ≥ Grade 2 lesions.

None of the medications significantly affected seroconversion or the incidence of IHC-positive pigs compared to the non-medicated control pigs.

Both the BMD plus Aureomycin and Tylan treatments showed significant reductions in faecal shedding (Figure 6).

A partial economic analysis was conducted using the following assumptions: Tylan at $0.13/g, Lincomix at $0.20/g, BMD at $0.06/g, and Aureomycin at $0.025/g. The medication cost/pig for the 25 day study was $0.213 for the BMD plus Aureomycin treatment, $0.263 for the Tylan treatment, and $0.411 for the Lincomix treatment (Figure 7).



Figure 1. Mortality rate of ileitis-challenged pigs fed three different medication programs or non-medicated


Figure 2. Average daily gain of ileitis-challenged pigs fed three different medication programs or non-medicated


Figure 3. Average daily feed intake of ileitis-challenged pigs fed three different medication programs or non-medicated


Figure 4. Feed/gain of ileitis-challenged pigs fed three different medication programs or non-medicated


Figure 5. Clinical evaluations of ileitis-challenged pigs fed three different medication programs or non-medicated.


Figure 6. Faecal shedding of ileitis-challenged pigs fed three different medication programs.


Figure 7. Treatment cost per pig of ileitis-challenged pigs fed three different medication programs.

Summary and Discussion

  • All three treatments prevented any PPE-related mortality, significantly reduced the signs of clinical ileitis, and gave reductions in the incidence and severity of intestinal lesions.
  • The BMD plus Aureomycin treatment showed the largest improvements in ADG, ADFI, and F/G, and significant reductions in faecal shedding.
  • Compared to the BMD plus Aureomycin treatment, the Tylan treatment cost an extra $0.05/pig and the Lincomix treatment cost an extra $0.20/pig.
  • BMD plus Aureomycin provided comparable or better ileitis control and better performance improvements than Tylan or Lincomix, and for less cost.

An ileitis control program utilizing BMD plus Aureomycin should be considered because of the synergies afforded by the combination, the flexibility and economics of usage, and the broad spectrum respiratory disease control provided. Only Alpharma CTC premixes (Aureomycin and ChlorMax) are approved for concurrent use with BMD. Continual use of BMD 30 g/ton in grower-finisher diets, coupled with strategically applied ‘pulses’ of Aureomycin at 10 mg/lb BW, will provide cost-effective ileitis control, performance enhancement, and broad-spectrum respiratory disease prevention/treatment, while allowing natural immunity to develop.

References

  1. Del Castillo, J.R.E. 1998. Metaphylaxis of bacterial respiratory problems. Pig Progress June issue.
  2. Del Castillo, J.R.E., et al. 1998. Pharmacokinetic modeling of in-feed tetracyclines in pigs using a meta-analytic compartmental approach. J Swine Health Prod 6(5):189-202.
  3. Feed Additive Compendium. 2008. p 224, 292, 447.
  4. Freese, W.R. 1991. Veterinarians on call. National Hog Farmer Jan.15:94.
  5. Gebhart, C.J. and S. McOrist. 1995. A hamster challenge model for evaluating effectiveness of chlortetracycline in prevention/control of porcine proliferative enteropathy. Proc Am Assoc Swine Pract p 93-99.
  6. Gebhart, C. 2000. Personal communication.
  7. Iowa State University Veterinary Diagnostic Laboratory 2007 Annual Report.
  8. Mathers, J.J. and V.R. Benning. 1998. Combination bactericidal activity of BMD and CTC against Clostridium perfringens of animal origin. CRWAD Proceedings p 8.
  9. McOrist, S., et al. 2000. Ileitis and Lawsonia intracellularis: what do we know today? Proc ISU Swine Disease Conf p 19-24.
  10. McOrist, S. and J. Morgan. 1998. An evaluation of chlortetracycline feed additive for control of porcine proliferative enteropathy (ileitis). Proc Int Pig Vet Soc 3:111.
  11. McOrist, S. and C.J. Gebhart. 1995. In vitro testing of antimicrobial agents for proliferative enteropathy (ileitis). J Swine Health Prod 3:146-149.
  12. McOrist, S. 1995. Proliferative ileitis: culture of etiologic agent, reproduction of the disease and sensitivity testing. Proc Am Assoc Swine Pract p 385-386.
  13. Opriessnig, T., et al. 2006. Chlortetracycline is effective in reducing lesions in pigs co-infected with Mycoplasma hyopneumoniae and porcine circovirus Type 2. Proc Int Pig Vet Soc 2:302.
  14. Thacker, E.L., B.J. Thacker, and T. Wolff. 2006. Efficacy of a chlortetracycline feed additive in reducing pneumonia and clinical signs induced by experimental Mycoplasma hyopneumoniae challenge. J Swine Health Prod 14:140-144.
  15. Thacker, E., D. Nilubol, and P. Halbur. 2006. Efficacy of Aureomycin chlortetracycline (CTC) granulated premix in decreasing the potentiation of PRRSV pneumonia by Mycoplasma hyopneumoniae. Proc Am Assoc Swine Vet p 153-156.
  16. Thacker, E. 2008. Comparison of Aureomycin chlortetracycline granulated premix to Lincomix in decreasing the potentiation of PRRSV pneumonia by Mycoplasma hyopneumoniae in an experimental model. Proc Am Assoc Swine Vet p 273-276.
  17. Veenhuizen, M.E., et al. 1998. The potential economic impact of porcine proliferative enteropathy on the US swine industry. Proc Int Pig Vet Soc 2:64.
  18. Walton, J.R. and J.E. Wheeler. 1987. Loss of resistance to the tetracyclines from porcine Escherichia coli in contact with dietary bacitracin methylene disalicylate. J Vet Med B34:161-164.
  19. Walton, J.R. and O.A. Laerdel. 1980. The effect of bacitracin in the feed on the resistance status of porcine strains of Escherichia coli. Proc Int Pig Vet Soc p 300.
  20. Walton, J.R. and R.G. Bird. 1975. A possible mechanism to explain the growth promotion effect of feed antibiotics in farm animals: zinc bacitracin induced cell wall damage in Escherichia coli in vitro. Zbl Met Med B22:318-325.
  21. Walton, J.R. 1975 (circa). The effect of bacitracin on the susceptibility of selected gram negative and gram positive bacteria to therapeutic antibiotics.
  22. Winkelman, N., et al. 1997. An evaluation of Aureomycin chlortetracycline (CTC) granular feed additive for prevention or treatment of swine ileitis. Proc Am Assoc Swine Pract p 79-83.
  23. Winkelman, N., et al. 2001. An evaluation of BMD® and ChlorMaxTM chlortetracycline (CTC) for control of challenge-induced porcine proliferative enteropathy (PPE or ileitis) in swine. Proc Am Assoc Swine Vet p 77- 82.
  24. Winkelman N., et al. 2003. An evaluation of BMD® plus Aureomycin® chlortetracycline (CTC), Tylan® or Lincomix® for control of challenge-induced porcine proliferative enteropathy (PPE or ileitis) in swine. Proc Am Assoc Swine Vet p 175-179.
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January 2009