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Control of Mycoplasma hyopneumoniae infection

Dr. Ulrich Klein, Novartis Animal Health Inc, Basel, Switzerland Dr. David Miller, Demafarma Consultancy Ltd., Walton on Thames, Surrey, England. Control programmes for M. hyo usually involve medication, management and vaccination.

M. hyo is sensitive in vitro to several antimicrobials such as tetracyclines, lincomycin, tylosin and the pleuromutilins. Table 1 summarizes the sensitivity results of M. hyo to various antimicrobials obtained in 5 independent studies dated from 1986 2002.

Although the MICs of tiamulin and several other antibiotics are low, for reasons that are obscure, pneumonia caused by M. hyo does not respond well to treatment (Ross. R.F., 1999). For example, Thacker, E. et al., (2000) reported that CTC was effective in preventing lesions and clinical signs associated with M. hyo when

such as chlortetracycline, doxycycline and oxytetracycline, against respiratory bacteria/mycoplasmal pathogens including M. hyo (see Table 3). The combination of tiamulin with a tetracycline provides truly comprehensive broad spectrum systemic activity which is particularly valuable in complex clinical

Table 1: In vitro sensitivity (mcg/ml) of M. hyo to various antibiotics (1986-2002)

Author Yamamoto et al. (86) Ter Laak et al. (91) Inamoto et al. (94) Hannan et al. (97) Thongkamkoon et al. (02) No. of strains tested 55 10 25 20 28 Linco. 0.12 0.25* 0.06 ND 0.097** OTC. 0.74 <0.03* 1.2 1.0** 0.39** CTC. 14.4 0.5* 36.4 ND 1.56 Tyl. 0.06 0.06* 0.03 0.25** ND Tia. 0.03 <0.03* 0.02 0.05** 0.048**

ND = Not determined *MIC50 = (minimum concentration required to inhibit 50% of the strains tested) **MIC90 = (minimum concentration required to inhibit 90% of the strains tested) Linco = Lincomycin OTC = Oxytetracycline CTC = Chlortetracycline Tyl = Tylosin Tia = Tiamulin

Field experience around the world for over 28 years since its introduction in 1978 has demonstrated that resistance to tiamulin among inherently sensitive organisms such as M. hyo develops extremely slowly, if at all. Hannan, P.C.T. et al., (1997) reported in a laboratory investigation that no significant resistance developed in M. hyo strains exposed to 10 in vitro passages in tiamulin containing broth. In the same study a high level of resistance to tylosin developed within 5-7 passages in tylosin containing broth (see Table 2).

administered in the feed prior to infection with v. However, CTC was ineffective when administered in feed on days 10 24 post infection. Muirhead, M.R. and Alexander, T. J. L. (1997) recommended that in the face of an outbreak, antibiotics with low MICs for M. hyo should be favoured, e.g. tetracyclines, lincomycin or tiamulin. A further significant property of the pleuromutilins is the pronounced in vitro synergism or enhanced activity, when combined with tetracyclines

situations of mixed microbial origin, e.g. porcine respiratory disease complex (PRDC) and post weaning multisystemic wasting syndrome (PMWS) or PRRSV infections. The new pleuromutilin derivative valnemulin also exhibits pronounced synergism against bacterial / mycoplasmal respiratory pathogens when combined with chlortetracycline and doxycycline [(Koh, H.B. et al., 2000) (see Table 6), Stipkovits, L. et al., (2004 (see Table 7)]. M. hyo is a key pathogen in the modern respiratory disease complex (porcine respiratory disease complex (PRDC) involving PRRS virus, PCV-2 virus and bacteria such as Actinobacillus pleuropneumoniae, Haemophilus parasuis, Streptococcus suis, Pasteurella multocida and Arcanobacterium pyogenes.

Table 2: In vitro development of resistance in M. hyopneumoniae strains (Type strain J,UK field strain MEVT 923)
Tiamulin Strain tested Pre-passage MIC (mcg/ml Post-passage MIC (mcg/ml) MIC increase J 0.1 0.25 2.5x Tiamulin MEVT 923 0.05 0.05 0 Tylosin J 0.25 >500 >2000x Tylosin MEVT 923 0.125 62.5 500x

Table 3: In vitro synergism between tiamulin and chlortetracycline (MICs in mcg/ml) against respiratory pathogens (Miller and Stipkovits, 1991, Burch et al. 1986, Jones, 1984)
Organism Tiamulin CTC Combined Tia / CTC Tia M. hyopneumoniae A. pleuropneumoniae (9) P. multocida (8) H. parasuis (1) B. bronchiseptica (1) 0.13 3.9 15.8 0.9 250 2.0 0.9 12.8 1.9 0.45 0.016 1.1 2.9 0.45 0.45 CTC 1.0 0.22 3.2 0.45 0.22 Tia 8x 3.6x 5.4x 2x 555x Synergy factor (x) CTC 2x 4x 4x 4.2x 2x

(strains tested)

Table 4: In vitro synergism between tiamulin and doxycycline against Mycoplasma spp. and respiratory bacterial pathogens (MICs in mcg/ml)* (Fodor, L. et al. (2004), Stipkovits, L. et al. (2004))
Organism Tiamulin Doxycycline Combined Tia / Doxy Tia M. hyopneumoniae A. pleuropneumoniae P. multocida S. suis B. bronchiseptica 0.219 2.297 2.297 0.094 16.0 5.169 0.435 0.125 0.189 0.088 0.094 1.071 0.870 0.044 5.656 Doxy 0.659 0.088 0.016 0.025 0.017 Tia 2.3x 2.1x 2.6x 2.1x 2.8x Synergy factor (x) Doxy 7.8x 4.9x 7.8x 7.6x 5.2x

10 strains tested

Table5 : MICs (mcg/ml) of tiamulin and oxytetracycline against 7 field isolates of M. hyopneumoniae (Koh, H.B. et al. 1994)
M. hyo isolate No. Tiamulin OCT Combined Tia / OCT Tia 1017 1034 2021 2022 2031 2039 2047 0.78 0.78 0.39 0.39 0.39 0.39 0.39 0.78 3.20 0.78 0.78 0.78 3.20 0.78 0.23 0.47 0.23 0.47 0.23 0.23 0.23 OCT 0.12 0.23 0.12 0.23 0.12 0.12 0.12 1.7x 1.7x 1.7x Tia 3.4x 1.66x 1.7x antagonism 6.5x 26.7x 6.5x Synergy factor (x) OCT 6.5x 13.9x 6.5x

Table 6: Synergistic in vitro activity (MICs mcg/ml) of valnemulin and chlortetracycline against respiratory pathogens (Koh, H.B. et al., 2000)
Organism Valnemulin CTC Combined Val / CTC Tia M. hyopneumoniae S. suis II P. multocida H. parasuis A. pleuropneumoniae 0.031 8 4 24 16 0.125 8 1 3 1 0.0012 0.0049 0.5-8 0.25-1 0.5-8 1-8 CTC 0.0098 0.0195 0.5-2 0.0625.0.25 0.0625-1 0.0625-0.5 Tia 25.8x/6.3x 16x/1x 16x/4x 48x/3x 16x/2x Synergy factor (x) CTC 12.8x/6.4x 16x/4x 16x/4x 48x/3x 16x/2x

Table 7: In vitro synergism between valnemulin and doxycycline against Mycoplasma spp. and respiratory bacterial pathogens (MICs in mcg/ml)* [Fodor, L. et al. (2004), Stipkovits, L. et al. (2004)]
Organism Valnemulin Doxycycline Combined Val / Doxy Val M. hyopneumoniae A. pleuropneumoniae P. multocida S. suis II B. bronchiseptica 0.120 2.143 1.741 0.023 11.31 5.169 0.435 0.125 0.189 0.088 0.101 0.870 0.435 0.011 4.287 Doxy 0.466 0.143 0.016 0.020 0.010 Val 1.2x 2.5x 4.0x 2.1x 2.6x Synergy factor (x) Doxy 11.1x 3.0x 7.8x 9.5x 8.8x

Table 8: Comparative efficacy of Tia/CTC vs tilmicosin against a mixed respiratory infection challenge (Stipkovits, L. et al. 2001)
Treatment Uninfected / Untreated Infected / Untreated Tia / CTC Tilmicosin Gain (kg) 10.0 8.85 10.1 9.95 Index (%) 100 88.5 101 99.5 FCR 1.93 2.44 1.90 1.88 Index (%) 100 126.4 98.4 94.4 Pneumonic lesion score 0% 100% 24% 40% Isolation M. hyo 0 / 10 8 / 10 0/10 3 / 10

The mycoplasmal and bacterial components are best controlled with the comprehensive broad spectrum antimycoplasmal / antibacterial activity provided by the combination of a pleuromutilin antibiotic (tiamulin or valnemulin) and a tetracycline antibiotic (CTC, OTC or doxycycline). Recent experimental work (Stipkovits et al., 2001) has clearly demonstrated the efficacy of Tiamutin/CTC (100/400ppm) in-feed medication against mixed M. hyo, Pasteurella multocida (Pm) and Actinobacillus pleuropneumoniae (App) infections, in comparison with tilmicosin (300ppm). 5-6 week old specific pathogen-

free piglets were experimentally infected with M. hyo on day 1, on day 8 with Pm and on day 15 with App. The premix treatment started on day 9 and continued for 12 consecutive days. The piglets were then autopsied for examination of macroscopic, histological and pathological lesions and for the presence of mycoplasmas and bacteria in the lungs. The Tia/CTC group showed an excellent response in average gain (+14.1%) and feed conversion efficiency (22.1% improvement) in comparison with the untreated, infected controls. When the lungs were examined for the presence of M. hyo it was not

possible to recover M. hyo from the Tia/CTC medicated group. However M. hyo was not eliminated from the lungs of the tilmicosin medicated group (see Table 8).

PULSED dosage programmes Pulsed in-feed dosage programmes ultilizing the Tiamutin/chlortetracycline or doxycycline combination can often be effective and economical in controlling: Mixed respiratory infections Combined respiratory / enteric infections In growing/fattening pigs, Guilmoto, H. (1996) reported on experience in Brittany, France on a breeder/fattener farm affected with mycoplasmal/bacterial pneumonias, which responded well to a pulse dosing programme with Tiamutin/CTC premix. Management changes, hygiene and environmental adjustments had previously failed to control the problem. The dosing protocol used was as follows: Tiamutin 80ppm + CTC 600 ppm (followed later by 400ppm). The pulse, consisting of Tiamutin + CTC commenced at 10-12 weeks of age and continued until 3 weeks prior to slaughter at 80kg bodyweight (bwt). The pulses were provided for 2 consecutive days and 8 x 2 day pulses were given in total. Over a 4 year period the percentage of lungs at slaughter which were free from pneumonia lesions increased from 10.66% in 1990 to 92.85% in 1994. Antibiotic treatments by injection became progressively less necessary and were finally phased out.

construction of building or pen manure handling feeding techniques access to water ventilation draughts bedding floor light heating hygiene characteristics of manager time dependent management factors (weaning, moving) movement of animals veterinary consultation temperature humidity gases bio-aerosols dust season distance to farm possibly infected size of neighbouring farm population pig population density in the region

associated with PRRS, A. pleuropneumoniae, influenza or pseudorabies heavy bacterial challenge necessity for continuous in-feed (antimicrobial) medication variable and poor growth associated with respiratory disease mortalities of more than 4% during the period weaning to slaughter cost of vaccination equal to or less than costs of associated mortality and in-feed medication Clark, L.K. (2000) in U.S.A. has proposed a simplified guideline: if a herd is positive for M. hyo and the pigs do not reach 115 kg in body weight by 180 days of age vaccination should be considered. The available scientific literature is not completely clear-cut on the optimal vaccination schedules. Few studies have tested the same vaccine in pigs of different ages on the same farms and their results do not all demonstrate marked performance differences (Desrosiers, R. 2001). However there is a tendency for vaccination performed later to produce better results [Jayappa, H. et al., (2001), Bilic, V. et al., (1996), Moore, C. and Daigneault, J. (2000) and Hodgins, D.C. et al., (2002)]. The time at which pigs are infected with M. hyo may be determined by serology, the time of onset of clinical signs and PCR and vaccinations may be scheduled in accordance with this information. Sorensen, V. et al., (1997) reported coughing approximately 2 weeks postinfection with M. hyo and sero-conversion approximately 1 week later. However Sorensen, V. et al., (1993) also warned that in field situations the interval between the introduction of nave pigs into an M. hyo infected population and the onset of clinical signs and sero-conversion may vary. Certain vaccines have been approved for use as single dose applications and therefore reduced handling costs. Yeske, P. et al., (2000) reported on a large scale US study which produced statistically and economically significant changes in performance when single dose vaccinated pigs were compared to non-vaccinated pigs.

Management interventions are often important in the toolbox of solutions required to successfully combat M. hyopneumoniae. Strk, K.D.C. (2000) has listed the following 33 risk factors reported to have an impact on respiratory disease: herd size air volume shared airspace stocking density diarrhoea sow characteristics herd type (breeding, fattening) purchase policy production system (all in, all out, batch, continuous)

M. hyo vaccines have now been in use around the world for several years and can reduce pneumonia and the financial losses associated with M. hyo infection. In severely affected herds a benefit : cost ratio of up to 5:1 has been achieved (Muirhead, M.R. and Alexander, T.J.L. 1997). However vaccination is not always an appropriate policy option and Muirhead and Alexander (1997) also suggested the following nine criteria to justify a decision to vaccinate: presence of M. hyo infection in the herd continuous level of respiratory disease presence of primary or secondary infections

An important question relating to M. hyo vaccination is whether or not sows should be vaccinated before farrowing. The evidence either way appears to be equivocal. A study by Thacker, B. et al., (2000) showed that piglets retaining maternal immunity had much less severe lung lesions postchallenge than piglets born from nave sows. However it was reported by Jayappa, H. et al., (2001) that piglets from vaccinated sows developed lower antibody titres when vaccinated whilst still retaining maternal immunity. Two reports which have compared the degree of protection to M. hyo challenge afforded by vaccines given to pigs with different levels of maternal immunity are Thacker, B.J. and Thacker, E.L. (2001), Jayappa, H. et al., (2001). In the first study there was no difference in the degree of protection afforded by the vaccine whether pigs were from vaccinated or unvaccinated sows. The vaccinated sows had been vaccinated pre-partum and the herd was M. hyo free. In the second study piglets from a naturally infected herd with high serum levels of antibody at the time of vaccination were not protected as well from M. hyo challenge at 16 weeks of age, as piglets vaccinated later when maternal antibody levels were lower. The authors concluded that when evaluating whether or not maternal antibodies might interfere with M. hyo vaccination of piglets, the actual levels of maternal antibody present in piglets at the time of vaccination could be of more significance than the age of the piglet. This underscores the importance of the assessment of the actual levels of maternal antibody present in piglets at the proposed time of vaccination. A possible complication of M. hyo vaccination is the interaction of M. hyo with concurrent PRRS virus infection. Vaccination with a modified live virus vaccine or natural infection with PRRS virus, during or following vaccination with M. hyo, appears to decrease the efficiency of M. hyo vaccination (Thacker, E.L. 2001). A further possible complication was described by Opriessnig and others at the IPVS Congress in Hamburg 2004. They showed that in concurrent infection with both PCV2 and M. hyopneumoniae severe

respiratory disease and lesions consistent with PRDC and PMWS occur. Further M. hyo vaccination enhances the clinical signs, macroscopic and microscopic lesions associated with PCV2 infections (Allan et al., (2000). The protection induced by M. hyo vaccines has been demonstrated clinically with both one and two shot vaccines, but infection is not prevented and only slight protection against M. hyo colonization is provided (Thacker, E.L. 2001). Vaccination of 1 week old piglets with an M. hyo vaccine significantly reduced clinical symptoms and lung lesions in piglets. However only a limited and non significant reduction in the transmission of M. hyo could be achieved. The authors noted that vaccination alone with the current bacterin vaccines will likely not be sufficient to eliminate M. hyo from infected herds. (Meyrs, T.B.S. and others 2006). US workers reported that vaccination against M. hyo in gilts did not prevent infection and infected incoming gilts, whether vaccinated or unvaccinated, are likely to shed M. hyo. (Pieters, M. and others 2006). M. hyo disease is dynamic and if an M. hyo vaccine is to be used the farm veterinarian must select a programme designed to suit the particular situation, based on the season, the production system and the presence of concurrent infections, both bacterial and viral. Vaccination against M. hyo, on account of the limitations described, is unlikely to be the optimum method to achieve eradication. Procedures to eliminate M. hyo and to produce M. hyo-free pigs using tiamulin Three different principles exist for the elimination / eradication of infectious diseases at herd level. These are: Total depopulation followed by restocking with non-infected animals. This method is widely used in Denmark to support the Danish SPF (specific pathogen free) scheme. Use medicated early weaning technique. Eradication without total depopulation and restocking, including temporary changes in production flow and use of

strategic antibiotic medication with a potent anti-mycoplasmal agent. This method is an alternative to restocking with SPF animals, is less expensive due to reduced loss of production and the genetic potential of breeding animals is preserved. Partial depopulation is only relevant for farrow to grower or farrow to finish herds and is a comparatively simple procedure to eradicate M. hyo. It was described originally by Zimmermann, W. et al., (1989) in Switzerland and has been tested with success in Denmark and Norway. The recommended procedure is as follows: All pigs (piglets, weaners, growers and finishers) younger than 10 months should be removed from the farm and there should subsequently be no farrowing during a 2 week period. For a 14 day period only breeding animals (sows/boars) older than 10 months must be present in the herd. Medication with tiamulin: during the 14 day period all breeding pigs are medicated with tiamulin either in feed or drinking water with 6-8 mg tiamulin per kg bodyweight. All units and pens are cleaned and disinfected. In Denmark modifications of the above general procedure have been carried out. The most common change is to exclude the cessation of farrowings and instead to inject all piglets in farrowing crates with Tiamutin injectable at dose of 10g thf per kg bodyweight on days 1, 7 and 14 of life. The relevance of the age limit of 10 months originally specified by Zimmermann has also been discussed in Denmark and a few herds have had success with a lower age of 8-9 months of age. Also in regard to younger breeding pigs of high genetic potential, movement to other premises and medication with tiamulin when they have reached 10 months of age has occurred. After medication they have

been returned to their herd of origin. The successful application of the above system has recently been confirmed in a report by Damgaard, K. et al., (2000). Clinical trials were carried out in Denmark on two herds acutely infected with M. hyo. Both herds were newly established SPF herds and were both re-infected by incoming gilts from the same breeding SPF herd which had been infected with M. hyo just before the gilts were sold. Herd 1 The herd was established in September 1992 with 180 gilts. In November 1992 gilts from the M. hyo infected herd were introduced. In February 1993 clinical signs were seen and 8/10 blood samples showed antibodies to M. hyo. In March 1993 an eradication programme using tiamulin was started. Herd 2 The herd was established in April 1993 with 360 gilts, some infected with M. hyo and some not infected with M. hyo. In August 1993 an eradication programme using tiamulin was started. In both herds the diagnosis was confirmed on the basis of clinical signs and a highly sensitive specific monoclonal blocking ELISA. The eradication programme was carried out in 2 phases: Phase 1: It was necessary at first to reduce the excretion of M. hyo from the acutely infected gilts. Thus all animals in herd 1 were medicated orally in the feed with tiamulin at approximately 8.0 mg thf/kg bwt for 14 consecutive days. In herd 2 only gilts with signs of enzootic pneumonia were medicated with tiamulin in drinking water at 8 mg thf/kg bwt for 7 consecutive days. Phase 2: In both herds gilts between 4 and 11 months of age were gradually divided into two groups. Animals over 10 months were totally separated from younger gilts in the cleaned/disinfected area during the following 3/4 months. In herd 1 both young and old gilts stayed in the same units but were kept in isolated, different rooms. In herd 1 gilts of >10 months were medicated, via feed with tiamulin at a dosage of 8.0 mg/kg bwt/day for 14 consecutive days.

In herd 2 gilts were medicated for 14 consecutive days with tiamulin; for the 1st 7 days in water and for the 2nd 7 days in the feed, with the same dosage of 8.0 mg/ thf/kg bwt/day. This process was repeated stepwise. Thus when the group of younger gilts reached the age of approximately 20 months they were again separated from the younger gilts and moved to another room and medicated with tiamulin as described above. The eradication programme was completed after 3-4 months when all remaining gilts has reached approximately 10 months and were medicated with tiamulin orally for 14 consecutive days. Additionally, in this transition period in herd 1 the piglets born were medicated on days 2 and 10 post partum with tiamulin injection (10 mg/thf/kg bwt i.m.). During this period internal barriers between medicated older gilts and younger non-medicated gilts were established to avoid the transmission of M. hyo infection from the younger gilts to the older tiamulin medicated animals. Separate sites of entrance to the different areas were established during the eradication period and personnel were not allowed to travel from the non-medicated group area to the medicated group area. Monitoring of both herds with clinical and serological tests was carried out posteradication. The eradication programme using tiamulin was considered by the authors to have been very successful. Both herds achieved SPF status again in Denmark after 1 year of blood testing. In herd 1 all blood samples were free fro M. hyo antibodies

Thus it was demonstrated that it was possible to eradicate M. hyo from acutely infected herds. Tiamulin medication of all animals in the acute phase was important to reduce the excretion of M. hyo (phase 1). Later the stepwise strict isolation of younger gilts from older gilts >10 months old permitted the eradication of M. hyo from the herds, even though the animals were maintained at the same farm. Christiansen, S. and Szancer, J. (2006) also described the successful elimination of M. hyo from a herd co-infected with PRRS virus and Actinobacillus pleuropneumoniae (Serotype 2, App 2). The farm had 600 breeding animals, sucklings and weaners up to 30kg b.w. and had an annual production of approx 14,000 x 30kg pigs. Diagnosis was based on serological tests, clinical signs and by culture of App 2. Mycoplasmal and bacterial isolates were tested in vitro and found to be sensitive to both tiamulin and chlortetra-cycline. On the day that medication was started there were no suckling pigs and all weaners were removed from the farm. There was no farrowing in the 14 day medication period and there were only breeding pigs >10 months of age on the farm. All breeding animals were treated via feed with a combination of tiamulin 3.5mg / kg b.w. (300ppm) and chlortetracycline 6mg / kg b.w. (500 ppm) for 14 consecutive days. On day 7 all breeding animals were injected once with marbofloxacin I/M at 4mg / kg b.w. The units with animals were cleaned and

Post-eradication period

Antibody to M. hyo No. of examined pigs Positive / negative 0 / 1680 0 / 480

Herd 1 Herd 2

7 2

for 7 years. In herd 2 all blood samples were negative for M. hyo for 2 years, but subsequently the herd was infected with A. pleuropneumoniae and restocked. Serological (ELISA) monitoring of herd 1 and herd 2 post-eradication programme for M. hyo infection was carried out:

disinfected daily for 14 days with a 2% solution of Virkon 5. Manure canals were emptied and disinfected just prior to eradication. Tools and boots were also cleaned and disinfected. The units emptied of animals were cleaned and washed with a glutaraldehyde

containing product and after disinfection the units remained empty for 2-3 weeks. After the eradication programme had been carried out the farm was monitored monthly by serological testing for 16 months for the presence of antibodies to M. hyo, App 2 and PRRS virus. During the 16 month period no positive samples for M. hyo (0/320) and PRRS (0/160) were found. One year prior to the eradication programme the average daily gain was 395g whilst one year post eradication it was 466g, a 17.9% improvement in performance. Successful attempts to eliminate M. hyopneumoniae without restocking (Lorenzen, J.B. 2000, Baekbo et al., 1994, Lium B. et al., 1992) and using the medicated early weaning method (Plomgaard, J. et al., 1992, Meszaros, J. et al., 1985) by medication with tiamulin were reported previously.

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