Denagard review of product characteristics and efficacy against enteric and respiratory infections

Ulrich Klein Novartis Animal Health Inc., Basel, Switzerland

Denagard is a pleuromutilin antibiotic developed, manufactured and marketed exclusively for animal health. Tiamulin, the active substance in Denagard, is a semi-synthetic antibiotic of the diterpene group. It exists in two forms. As tiamulin hydrogen fumarate which is used in all oral formulations (premix, water-soluble formulations). Tiamulin base is used in the injectable form.

Pharmacokinetics
Tiamulin is well absorbed in the pig (> 90%) and widely distributed following oral administration. Plasma levels are readily achieved after a short period of time. Tiamulin penetrates into cells (enterocytes, lung epithel cells) and accumulates inside them. Metabolism takes place in the liver the metabolites do not possess antimicrobial activity so all clinical activity is related to the parent compound. Tiamulin has been shown to preferentially concentrate in the lung tissue due to its pKa and lipophilic properties. Numerous publications have shown that at control and treatment dose levels lung tissue concentrations can be expected to exceed the MICs for bacterial respiratory pathogens.

Antimicrobial activity
Tiamulin is highly active against Mycoplasma, Brachyspira and Lawsoniaall being important aetiological agents in pig diseases. Tiamulin is also highly active against Staphylococci, Streptococci, Clostridium perfringens and, to a lesser extent, Listeria monocytogenes and Erysipelothrix. Tiamulin provides antimicrobial activity against a number of Gram-negative respiratory pathogens such as Actinobacillus spp., and Pasteurella multocida. Minimum inhibitory concentrations (MICs) of tiamulin against a wide range of bacterial organisms are determined continuously and the activity of tiamulin

Mode of Action
The mode of antimicrobial action is via inhibition of protein synthesis by targeting the large subunit (50s) of the bacterial ribosome, thus exerting an inhibitory effect upon growth. Denagard is bacteriostatic in action.

Table 1: Sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli field strains to tiamulin (MICs in mcg/ml)
Reference Aitken et al., 99 Moller et al., 96 Karlsson et al., 01 Karlsson et al., 01 Luengyosluechakul et al., 02 Karlsson et al., 04 Rohde et al., 04 Reference Fellstrm et al., 96 Dalziel 1998 Moller 1998 Kinyon et al., 02 Cizek et al., 04 VLA 03
n.d. = not determined

No. B. hyodysenteriae isolates 10 10 50 25 7 20 102 No. B. pilosicoli isolates 10 12 5 25 10 20

MIC range / mean 0.025-1 0.125-1 0.031-2 n.d. 0.125-4 mean: 0.129 mean: 0.129 0.031-8 MIC range / mean 0.0125-0.5 0.031-0.05 0.0156-0.0625 0.06-8 0.03-1.0 mean: 0.148

MIC50 0.3 0.125 0.125 0.125 n.d. n.d. 0.5 MIC50 n.d. 0.067 0.041 0.125 0.250

MIC90 1 1 0.125 1.0 n.d. n.d. 2.0 MIC90 n.d. n.d. n.d. 1.0 1.0

is compared with that of other therapeutic drugs. Tiamulin possesses high in vitro activity against Brachyspira hyodysenteriae (B. hyo), the organism resposible for swine dysentery and Brachyspira pilosicoli (B. pilo) the causative agent of Porcine Colonic Spirochaetosis (PCS). MIC studies conducted worldwide indicate a high susceptibility of B. hyo and B. pilo field strains (Table 1). Based on the breakpoints described for tiamulin by Ronne and Szancer (1990; sensitive <1 / intermediate >1-<4 / resistant >4) the results of the laboratory investigations summarized in Table 1 demonstrate a high sensitivity of B. hyo and B. pilo field strains isolated worldwide. Denagard has been shown to be highly effective against Lawsonia intracellularis (L.i.) using intracellular and extracellular assays (Table 2). It is particularly suitable for the effective control of L.i.-infections as it concentrates inside the cells where the organism lives. Tiamulin has proven synergistic activity against important respiratory pathogens when used in combination with Tetracyclines (chlortetracycline, oxytetracycline, doxycycline). Data published by Fodor et al., (2004) show the synergistic effect of Denagard/doxycycline combinations. The combined use provides broad spectrum activity which is particularly valuable in the case of the treatment of complex respiratory infections.

Table 2: Sensitivity of Lawsonia intracellularis strains to tiamulin (McOrist et al. 1995) MICs in mcg/ml
Antimicrobial Tylosin Lincomycin Spectinomycin Tiamulin No. of strains tested 3 2 1 3 MIC intracellular activity 64 32 32 4 MIC extracellular activity 64 32 32 4

Efficacy against Swine Dysentery

Swine dysentery (SD) remains a major health issue in many pig-rearing countries in Europe, Asia/Pacific, Latin America as illustrated by recent reports of the disease from Brazil (Barcellos et al., 2000), Thailand (Prasasarakul et al., 2004), Japan and several European countries (Lobova et al., 2004, Rasbaek et al., 2003). The incidence of the disease is currently increasing primarily in Europe (Merialdi 2005, Raesbaek 2004). Control and therapy of the disease, caused by Brachyspira hyodysenteriae (B. hyo), is based on the administration of antimicrobials. No effective vaccine is available on the market. Denagard possesses exceptional antimicrobial potency against B. hyo and is also effective against other anaerobes (Fusobacterium spp., and Bacteroides spp.,) which contribute to the development of the gut lesions of swine dysentery. Denagard is effective against B. hyo strains,

which have become resistant to lincomycin, tylosin. Several studies have been reported, among others, from Poland, Sweden, UK and Australia which confirm this specific effect. In some herds swine dysentery is diagnosed regularly and is considered to be the infection that causes most losses. In those cases its important to eliminate the infection from the herd. Due to the considerably lower costs medical eradication programmes are more often used than converting to specific pathogen-free (SPF) production by re-stocking. There is a good correlation between the in vitro effect (MIC values) of the antimicrobials and their ability to eliminate B. hyo from pigs. Denegard is able to eliminate Brachyspira hyodysenteriae and is used as the drug of choice for eradication programmes. Recently data on Denagard eradication programmes in big farms (2000 and 6000 sow level) in Australia by partial depopulation were published by McOrist

Table 3: Sensitivity of respiratory pathogens and in vitro synergism between tiamulin and doxycycline (Fodor et al., 2004) MICs in mcg/ml
Organism M. hyopneumoniae M. hyorhinis M. hyosynoviae A. pleuropneumoniae P. multocida Strep. suis B. bronchiseptica Tiamulin 0.219 0.219 0.12 2.297 2.297 0.094 16 Doxycycline 5.169 0.933 1.101 0.435 0.125 0.189 0.088 Combined Tiamulin 0.094 0.116 0.044 1.071 0.87 0.044 5.656 Doxycycline 0.659 0.094 0.116 0.088 0.016 0.025 0.017 2.3x 1.9x 2.7x 2.1x 2.6x 2.1x 2.8x Synergy Factor Tiamulin Doxycycline 7.8x 9.9x 9.5x 4.9x 7.8x 7.6x 5.2x

and Bennet (2006). The farrowing rates in both farms and the number of piglets born alive were improved post-eradication over pre-depopulation levels on an incremental basis (See Table 4). At each site the farrowing rate increased 3% and the number of pigs born alive increased 0.9 to 1.5 above baseline levels within six months. The published data illustrate that eradication of swine dysentery is possible not only in small but also on large units.

Efficacy against Porcine Colonic Spirochaetosis (PCS, colitis)

Colitis is an ongoing problem in pig herds worldwide. Surveillance data confirm that Brachyspira pilosicoli is the most prevalent cause of this infection. Although Porcine Colonic Spirochaetosis is a common problem, there have been few farm-based trials to assess the cost of the disease and the cost-benefit of treatment and prevention programs. One trial reported

improvements of 18% in average daily gain (ADG) and 0.14 in feed conversion ratio (FCR) in a batch of weaners treated with Econor in feed for 28 days (Glossop et al., 2000). To provide further informations on the cost benefits of controlling PCS, a trial was conducted over the course of a year in a commercial grower unit, where PCS had been a repeated problem. The trial results were reported by Thomson et al., (2006). Pigs were farmed within a minimal disease pyramid that was free from all serious endemic diseases (PRRS, EP, APP, PAR, S. suis II, SD, and mange). Pigs were delivered from the nursery unit at approximately 9 weeks of age (approximately 25kg) and remained at the trial farm for 6 weeks. Pigs were weighed on arrival and divided between 4 large straw-bedded pens in one large shed. Pigs in two pens received feed medicated with Denagard (100g tiamulin hydrogen fumarate per tonne feed) for the first 7 to 10 days, and thereafter received non-medicated feed. After 6 weeks the pigs were weighed then transported to the finisher unit. Between batches the building was emptied and cleaned, giving a between-batch interval of 2 weeks. Six sequential batches of pigs were tested over the course of one year. Denagard was used as B. pilosicoli isolates from the unit showed high sensitivity to tiamulin during preparatory studies. The data are showing that Denagard effectively reduced the clinical expression of B. pilosicoli infection. PCS-infection in non-medicated pigs led to significantly poorer daily weight gain (average 14%) and feed conversion ratio (average 12.8%) as compared with the Denagard treated pigs. The cost benefit evaluation demonstrates the

Table 4. Performance results pre-and post-eradication in farms in Australia (McOrist and Bennet, 2006)
Preeradication Farrowing rate Pigs born alive per sow Average daily weight gain (kg/day wean-to-finish) 78-80 9.5-10.6 0.63-0.73 Posteradication 80-84 10.4-11.3 0.68-0.76 Improvement 3% 10% 10%

Table 5. Comparative data for Tiamutin treatment versus no treatment to prevent B. pilosicoli infection in a commercial grower unit (Thomson et al., 2006)
No treatment (control) Batch No. pigs ADG (g/day) 1 2 3 4 5 6 300 305 315 300 310 305 725 750 760 820 780 705 FCR 2.0 1.9 2.1 2.3 1.9 2.0 Treatment (Tiamutin) No. deaths 3 5 2 1 4 2

No. deaths No. pigs ADG (g/day) FCR 2 3 6 2 4 3 310 320 310 300 306 310 840 838 920 950 810 920 1.8 1.7 1.8 1.9 1.7 1.7

Table 6. Statistical comparison of treatments (using paired t-test on batch means)

No treatment ADG (g/day) FCR Mortality (%) 757 2.03 1.08 Tiamutin 880 1.77 0.91 sem 25.7 0.033 0.262 Significance P=0.005 P<0.001 Not significant

Table 7. Cost / Benefit evaluation () based on mean data for the six batches as shown in Table 5
Estimated additional costs per control batch* Additional feed costs# Additional overhead costs Costs of Denagard per treated batch** Estimated benefit / cost ratio of controlling Brachyspira pilosicoli colitis with Tiamutin
* Estimated additional costs per batch for pigs in the control (untreated) groups to reach the average end weight of the treated batches (target end weight). # Cost of grower ration: 233 per tonne. Estimated 6 additional days on farm to attain target end weight. Based on British Pig Executive overhead cost figure of ?0.33 per grow-finish pig per day. ** Cost of Tiamutin medication: 36 per tonne feed.

665.35 600.00 143.0 8.8 : 1

mortality in any of the three groups, weight gain in group 3 (challenge controls) was reduced to 30% of group 1 (non-challenged controls) from day 7-22 (179 vs. 598 g/ hd/d). The administration of Denagard in the water did provide significant (p<0.05) improvement in the weight gain compared to group 3, but was significantly lower than group 1. The PPE challenge also significantly (p<0.05) reduced feed and water intake and negatively impacted feed conversion when comparing groups 1 and 3. The administration of Denagard significantly (p<0.05) improved feed conversion when comparing groups 2 and 3. In a second study (Walter et al., 2001) the effectiveness of 35 g/t (38.5 ppm) Denagard in feed for control of PPE was evaluated. Forty-eight five-week-old pigs were randomized into 16 pens (3 pigs/pen) comprising of blocks of two treatments and given a pure culture Li challenge on Day 0. Nine days later when > 50% of pens had pigs with diarrhoea, Denagard medication was started and continued for 28 days in group 1. Group 2 served as Li challenge controls. Both groups were necropsied to analyze tissues for gross and microscopic PPE lesions. The clinical signs were significantly reduced

economical value of Denagard medication against Brachyspira pilosicoli-associated field infection in this unit. The benefit cost ratio of 8.8:1 of controlling colitis with Denagard is related to the grower stage and does not take into account the possible ongoing adverse effects of B. pilosicoli infection in the finisher unit. The cost of disease up to finishing could be higher than indicated in this study.

obtained from the ileum of a breeding stock gilt with Porcine Haemorrhagic Enteropathy (PHE). Seven days following Li challenge when > 15% of the pigs had diarrhoea, Group 2 was administered tiamulin hydrogen fumarate (Denagard) at 60 ppm in the water for five days. Group 3 received nonmedicated water. Pigs were observed for a 10 day post-medication period. Individual body weights were recorded on days 0, 7, 12 and 22. The impact of PPE on growth performance was dramatic. Although there was no

Efficacy against Proliferative Enteropathy (PPE, Ileitis)

The efficacy of Tiamutin/Denagard against Porcine Proliferative Enteropathy has been evaluated in numerous field and challenge trials. The specific effect of Denagard on Lawsonia intracellularis was proven irrespective of whether it was administered in feed, in water or as an injectable. One trial was conducted to evaluate the effectiveness of Denagard 60 ppm administered in the water for five days for ileitis control using a Li mucosal homogenate challenge (Winkelman, 2002). Eighteen crossbred pigs 6 weeks of age (9.0 12.2 kg) were randomized by weight into three groups and placed into individual pens (n = 9). Group 1 received non-medicated water and served as nonchallenged controls. Group 2 and 3 were orally challenged on Day 0 with 1 x 109 Li per pig of a mucosal homogenate originally

Table 8. Performance Results Ileitis challenge study (Winkelman, 2002)

Treatment Groups Li Challenge Denagard Day 22 (necropsy) ADG (g) ADFI (g) ADG:ADF ADWI Mortality
P value < 0.05

Group 1 (n=9) No 0 ppm 22.86a 594a 1146a 0.518a 3116a 0/9

Group 2 (n=9) Yes 60 ppm 18.42b 402b 908b 0.424a 2457b 0/9

Group 3 (n=9) Yes 0 ppm 15.16c 179c 855b 0.201b 2592b 0/9

Figure 1. Mean clinical symptom scores (Walter et al., 2001)

1.5 Mean Clinical Symptom Scores Initiation of treatment

Efficacy against Porcine Respiratory Disease Complex (PRDC)

The respiratory disease complex causes major economic losses to swine producers. One of the main etiological factors of this complex is Mycoplasma hyopneumoniae (M. hyo). M. hyo is considered to play a major role, along with PRRSv, swine influenza virus and other bacterial respiratory pathogens in porcine respiratory disease complex. Only a few data on testing drugs in a combined M. hyo + P. multocida + A. pleuropneumoniae infection model are available. Stipkovits and colleagues (2001) compared in a study the effect of a Denagard/ chlortetracycline combination with other therapeutics for the in-feed treatment of pigs experimentally infected with M. hyo + P. multocida + A. pleuropneumoniae. In the study 6-week old pigs were infected on day 1 with M. hyo, on day 8 with P. multocida and on day 15 with A. pleuropneumoniae. The following medications were tested: Group 1: Denagard 100ppm + CTC 400ppm Group 2: Lincomycin 100ppm + CTC 400ppm Group 3: Pulmotil 300ppm The treatment was initiated on day 9 and continued for 12 consecutive days. The animals were monitored on days 8, 15, 22 and were also evaluated upon necropsy. In the infection model involving M. hyopneumoniae, Pasteurella multocida and Actinobacillus pleuropneumoniae the efficacy of the therapeutics were judged by clinical, microbiological, pathological and productivity criteria. Animals medicated with Denagard/CTC showed the most

1.0 2 Died
Nonmedicated Tiamulin hfu

Day of Infection 0.5

0.0

-9

Feed study

7 Study Day

14

21

28

after 7-10 days on treatment. Faecal shedding of Li was significantly reduced after 14 days on medication. There was no effect on Li sero-conversion between treatments. ADG and G/F were significantly greater after 21 days on medication. The prevalence and severity of gross microscopic PPE lesions were significantly reduced in the 35g/t treatment group. The study demonstrates the ability of Denagard in feed to control PPE in pigs even when given after clinical signs have developed.

The

studies

show

that

Denagard

administered in the feed (35 g per ton) or in water (60 mg per liter) is very effective in treating and controlling Porcine Proliferative Enteropathy and minimizing the negative effect of PPE on growth performance. In 2006 cost benefit data from trials in Europe and Asia/Pacific were published which verify the economic value of the treatment of Porcine Proliferative Enteropathy with Denagard (Klein et al., 2006, Burch et al., 2006, Poolperm et al., 2006, Shimaoka et al., 2006)

Table 9. Performance Results Ileitis challenge study (Walter et al., 2001)

Non-Medicated n = 24 ADG (g) ADFI (g) ADG:ADFI Pigs with PPE gross lesions (%) Pigs with microscopic lesions (%) Pigs with faecal Li shedding(%) 409 760 0.54 33% 1146a 17% Denagard n = 24 528 840 0.63 4% 908b 0% P-Value

.007 .029 .01 .0206 855b .05

Table 10. Performance and clinical scores of infected pigs fed with premix formulations of different antibiotics (Stipkovits et al., 2001)
Treatment Weight gain in (kg) 8.85 Feed conversion 2.44 Occurrence of pneumonia 10 / 10 Isolation M.hyo 8 / 10 Isolation App 6 / 10

Infected untreated controls

Lincomycin/ CTC

8.65

2.23

6 / 10

5 / 10

2 / 10

Pulmotil

9.95

1.88

5 / 10

3 / 10

1 / 10

Denagard/CTC

10.1

1.90

2 / 10

0 / 10

1 / 10

Figure 2. Mean lung scores (Stipkovits et al. 2001)

58 44 80 Lung lesion score) 60 40 20 0 14

C o ntr o l Denagard/CTC L incomycin/C T C P ulmotil 23

pronounced reduction in the clinical signs, the lowest incidence of pathologic lung lesions, a major reduction in Mycoplasma contamination and the lowest occurrence of pneumonia. The satisfactory clinical efficacy of Denagard/CTC resulted in improved pig performance. The data from Stipkovits et al., confirm previously and recently published data which prove the synergistic effect of Denagard and tetracyclines when used together. Those efficacy data are based

on the specific molecular mode of action of both therapeutics. Denagard provides action towards the 50s ribosomal subunit and tetracycline`s action is toward the 30s ribosomal subunits. With the activity at these different ribosomal subunits it is easier to understand the synergy of Denagard and tetracyclines against bacterial pathogens which cause complex respiratory infections in pigs.

Efficacy against Actinobacillus pleuropneumoniae infection

Denagard administered both parenterally and orally accumulates in lung tissues in preference to serum since it is highly lipophilic. In experimental challenge studies published by Taylor (2004) the efficacy of Denagard was tested against Actinobacillus pleuropneumoniae (APP). Denagard was administered via water at a dose level of 120 ppm.

Table 11. Performance and clinical scores of infected pigs medicated with Denagard (Taylor, 2004)
Treatment Pig numbers Weight Gain Daily Water used Total feed intake Days App Days fever Mortality Lesion extent lung Plasma tiamulin Tiamulin normal lung Tiamulin pneumonic lung
n.d. = not determined

Non-medicated 6 3.5- kg 390 ml 8.0 kg 13/21 (38%) 12/24 (50%) 3/6 (50%) 38% n.d. n.d. n.d.

Denagard 6 0.5+ kg 1002 ml 17.0 kg 5/36 (14%) 13/42 (31%) 0 1.9% 0.0-0.2 mcg/ml 1.3-22.3 mcg/ml 5.0-15.5 mcg/ml

Tiamutin water medication (dosage 120 ppm) prevented mortality, reduced the clinical signs in established acute pleuropneumonia and prevented the persistence of lung lesions. Lesions of pleuropneumonia were markedly more severe in untreated pigs. MICs (3.0 mcg/ ml) of the inocular APP strain and all re-isolates showed high sensitivity based on the breakpoints for tiamulin and APP (sensitive <8.0; intermediate 9-16; resistant >16; Casals, 1990). The MICs were exceeded in both normal and affected lungs. These findings help explain field observations that Denagard is effective in treating pleuropneumonia (Anderson, 1986, Edwards et al., 1992 and Jordanov et al., 1996).

Pigs affected by pleuropneumonia might not eat normal amounts of feed. Based on the obtained results of the infection study it can be concluded that water medication with Denagard can treat pleuropneumonia successfully by preventing mortality and reducing production loss and lung lesions. APP field isolates from a number of countries have MICs within the range at which sensitivity to Denagard would be expected, suggesting that is could more be more widely used.

characteristics, high concentrations at the side of infection in the gut and in the lung are achieved. Trial data confirm the pronounced efficacy against key diseases in swine. Enteric, respiratory and complex mixed infections can reliably be controlled with Denagard which results in higher performance and an economical benefit for the producer.

Conclusions
Denagard provides antimicrobial potency against a broad spectrum of bacterial pathogens. Based on its pharmacokinetic

References
D.Burch et al., (2006). Comparative efficacy of Tiamutin and Linco-Spectin in the drinking water for the treatment of mixed enteric and respiratory infections in finishing pigs. Proceedings 19th IPVS Congress, Copenhagen, Denmark, Vol. 2, p343. L. Fodor (2004). Sensitivity testing of respiratory swine pathogens to antimicrobials. Proceedings 18th IPVS Congress, Hamburg, Germany, Vol. 2, p 563. U.Klein et al., (2006). A cost benefit study on the control of Porcine Proliferative Enteropathy in a commercial grower unit in Germany. Proceedings 19th IPVS Congress, Copenhagen, Denmark, Vol. 2, p167. S.McOrist and C.Bennett (2006). Eradication of swine dysentery on large-scale breeder farms by partial depopulation /medication. Proceedings 19th IPVS Congress, Copenhagen, Denmark, Vol. 1, p319.

P.Poolperm et al., (2006). Treatment comparison between Tiamutin and Tylan against mixed enteric infection with PRRS co-infection in Thailand. Proceedings 19th IPVS Congress, Copenhagen, Denmark, Vol. 2, p347. M.Shimaoka et al., (2006). Efficacy and cost benefit study on the use of Tiamutin for the treatment of Porcine Proliferative Enteropathy under field conditions in Japan. Proceedings 19th IPVS Congress, Copenhagen, Denmark, Vol. 2, p183. L.S.Stipkovits et al., (2001). Treatment of pigs experimentally infected with Mycoplasma hyopneumoniae, Pasteurella multocida and Actinobacillus pleuropneumoniae with various antibiotics. Canadian Journal of Veterinary Research, 65: 213-222. D.J.Taylor (2004). Treatment of Actinobacillus pleuropneumoniae (APP) infection by water medication with Tiamutin. Proceedings 18th IPVS Congress, Hamburg, Germany, Vol. 2, p 509.

J.R.Thomson et al., (2006). A cost-benefit study on the control of Porcine Colonic Spirochaetosis in a commercial grower unit. Proceedings 19th IPVS Congress, Copenhagen, Denmark, Vol. 2, p 350. D.Walter et al., (2001). Treatment and Control of Porcine Proliferative Enteropathy Using different Tiamulin Delivery Methods. J. Swine Health and Prod 9 (3): 109-115. N.Winkelman et al., (2002). The Impact of Tiamulin Administered in the water on the Performance of Growing Swine with Clinical Porcine Proliferative Enteritis 17th IPVS Congress, Aimes, USA, Vol.2, p 197.

Denagard is a registered trademark of Novartis AG, Basel, Switzerland. Pulmotil is a registered trademark of Eli Lilly and Company 2011 Novartis Animal Health Inc.