Avian Pathology (2003) 32, 213 /216
CASE REPORT
Apparent eradication of Mycoplasma synoviae in broiler breeders subjected to intensive antibiotic treatment directed to control Escherichia coli Laurimar Fiorentin1*, Ricardo A. Soncini2, Jose´ Luiz A. da Costa2, Marcos A. Z. Mores2, Iara M. Trevisol2, Ma´rcia Toda2 and Nilson D. Vieira1 1
Brazilian Agricultural Research Corporation EMBRAPA, Embrapa Suı´nos e Aves, BR 153, km 110, Vila Tamandua´, Caixa Postal 21, 89700-000, Conco´rdia, SC, Brazil, 2Sadia SA, Rua Senador Attı´lio Fontana 86, 89700-000, Conco´rdia, SC, Brazil /
A Mycoplasma synoviae (MS)-free flock of broiler breeders was housed for brooding and rearing on an MS endemic farm. PCR revealed that the flock became infected within nine weeks. At 22 weeks the flock was transferred to a clean and disinfected house on a previously depopulated farm. The birds were then subjected to three treatments with fluoroquinolones due to recurrent Escherichia coli peritonitis and from the 32 weeks of age they received 600 ppm of oxytetracycline hydrochloride continuously in the feed. Monitoring by PCR showed a decrease in MS positive birds after 34 weeks of age and MS may have been eradicated as judged by consistent negative results in PCR. We conclude that intensive antibiotic treatments supported by adequate biosecurity could clear MS from infected broiler breeders.
Introduction Mycoplasma synoviae (MS) infection is usually asymptomatic in broiler breeders in Brazil but the possibility that it might play an important role in complex respiratory disease in the offspring has motivated breeding companies to consider eradication. Broiler breeders are usually maintained under high biosecurity while broilers are often reared in areas where infectious bronchitis is endemic and Escherichia coli is a constant threat. In the past, mycoplasma eradication programmes have been based on antibiotic or heat treatment of fertile eggs. Mycoplasmas are sensitive to a number of different antibiotics including tetracyclines (Cerda et al., 2002; Chopra & Roberts, 2001) and fluoroquinolones (Cerda et al., 2002; Hannan et al., 1997; Jordan et al., 1998; Wellehan et al., 2001). These are broad spectrum antibiotics and can be used to control a wide range of bacteria including E. coli. Here we report the
fortuitous finding that three in-feed treatments of broiler breeders with fluoroquinolones, coupled with treatment with oxytetracycline, which were aimed at controlling recurrent E. coli peritonitis, also resulted in apparent eradication of MS from the flock.
Material and Methods Source of broiler breeders Broiler breeders were obtained from an MS- and Mycoplasma gallisepticum - (MG) free primary breeding stock farm in the south of Brazil. The mycoplasma-free status had been confirmed by the Brazilian Ministry of Agriculture by repeated serological testing by serum plate agglutination (SPA) and haemagglutination inhibition (HI) tests at 6 to 8 week intervals throughout the flock life, plus at least one attempt to isolate MS from tracheal swabs. In addition we carried out MS and MG polymerase chain reaction (PCR) on tracheal swabs from birds of three ages (19, 33 and 66 weeks) and confirmed the negative status.
*To whom correspondence should be addressed. Tel.: /49 442-8555; Fax: /49 442-8559; E-mail:
[email protected] Received 24 June 2002. Provisionally accepted 31 August 2002. Accepted 2 October 2002 ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/03/020213-04 # 2003 Houghton Trust Ltd DOI: 10.1080/0307945021000071641
214 L. Fiorentin et al. Housing and flock history The birds in this study were hatched at the grandparent company’s hatchery and brooded and reared on a high biosecurity farm with two 32 000 bird all-in all-out quarantine units. Asymptomatic MS infection, usually detected by serology and occasionally confirmed by isolation from tracheal swabs, had occurred in every flock housed in recent years on the farm. At 22 weeks the birds were transferred to a farm that had been depopulated, cleaned and disinfected. They were housed in four houses and 12 000 of one house formed the population for the continued study. After 26 weeks the flock showed recurrent E. coli peritonitis and received several in-feed antibiotic treatments. These included phosphomycin (PHO; Ilender do Brasil), enrofloxacin (ENR; Fatec SA) and norfloxacin (NOR; Formil Quı´ mica LTDA) (Table 1). From weeks 32 to 59 in-feed oxytetracycline hydrochloride (OXT) (Huashu Pharmaceutical Corp.; 600 ppm) was also given continuously. At 67 weeks the flock was slaughtered but 10 hens were retained and housed in isolators in two groups of five. One group was vaccinated by eye-drop with infectious bronchitis virus vaccine (Broilerbron-H120; Schering-Plough Coopers) at 10 times the recommended dose in an attempt to exacerbate any MS infection. All 10 birds were necropsied at 69 weeks.
Sampling and laboratory tests Ten tracheal swabs for PCR and 10 more for mycoplasma isolation were collected at 9, 15, 23, 29, 34, 47, 54 and 59 weeks of age together with 20 blood samples. PCRs were carried out on individual swabs by the method of Lauerman et al. (1993). Mycoplasma isolation was attempted for each swab in 3 ml of broth (Frey et al. , 1968) supplemented with 0.1 g/l nicotinamide adenine dinucleotide and 0.1 g/l cysteine hydrochloride. Broths showing pH change or turbidity were plated onto agar (Frey broth with 0.75% agarose). All other broths were plated out weekly for three weeks. Mycoplasma isolates were identified by immunofluorescence and filter-cloned three times (Bradbury, 1998). SPA tests were performed on the farm on fresh sera using commercially prepared antigen (Intervet B.V., Boxmeer, the Netherlands) and the HI test (Kleven and Mycoplasmosis, 1998) was carried out on the serum collected at 29 weeks.
Table 1.
Tracheal swabs for PCR and culture and blood samples for RSA and HI tests were also collected at 67 and 69 weeks from the 10 birds that were moved to isolators when the flock was slaughtered. These birds were necropsied at 71 weeks and culture and PCR was attempted from trachea, airsacs and hock joints. Internal organs and hock synovial membranes were examined for histological lesions (Luna, 1968). Minimal inhibitory concentrations (MICs) for ENR, NOR and OXT were determined in vitro (Hannan, 2000) with the type strain WVU1853 and with two MS isolates (443 and 563) from the previous batch of birds on the same brooding and rearing farm. PHO was not included as its mode of action is to inhibit peptidoglycan synthesis and therefore it has no activity against the cell wall-less mycoplasmas.
Results PCR indicated that the MS-free flock that was placed on the MS endemic site was positive by 9 weeks of age and 70% or more swabs were PCR positive up to 29 weeks (Table 1). After treatments had commenced these fell to 1 in 10 PCR positive swabs by 34 weeks, thereafter there were no further positive PCR reactions. SPA reactions were first seen at 29 weeks and persisted until 59 weeks. By the time the flock was slaughtered at 67 weeks there were no positive SPA reactors and the 10 hens that were retained, including the five that received live IBV vaccine, remained PCR and SPA negative. No positive HI reactions were recorded during the entire study nor were signs or lesions related to MS seen in either the flock prior to slaughter or in the 10 retained birds. No microscopic lesions of airsacculitis or synovitis were seen in the tissues taken from these 10 birds. MIC breakpoints were 2 mg/ml for OXT, 0.125 mg/ml for NOR and 0.25 mg/ml for ENR while the
Timetable of events and laboratory results for the flock from 0 to 69 weeks of age Positive for MS by
Week 0 9 15 22 23 26 28 29 32 34 39 47 52 54 59 67 69
a
PCR (of 10) MS-free flock housed on MS endemic farm 7 10 Flock moved to a clean farm 9 E. coli peritonitis diagnosed; 35 mg/kg phosphomycin in feed for 5 d 10 mg/kg enrofloxacin in feed for 5 d 10 600 ppm oxytetracycline started in feed (continuous) 1 10/ mg/kg enrofloxacin for 5 d 0 10 mg/kg norfloxacin for 5 d 0 0 Oxytetracycline withdrawn Flock slaughtered; 10 hens moved to isolators; 5/10 given 10 / IBV 0 0 Remaining 10 birds necropsied; no lesions seen
out of 20 birds. out of 10 birds.
b
Isolation (of 10)
RSA (of 20)
HI
0 0
0 0
nd nd
0
0
nd
0
20
0a
0
20
nd
0
20
nd
0 0
20 20
nd nd
0 0
0b 0b
0b 0b
Mycoplasma synoviae eradication
breakpoints for the type strain were 2 mg/ml for OXT and ENR and 1 mg/ml for NOR. All these were considered to be in the sensitive range.
Discussion It has been reported that MS strains can differ in virulence (Fiorentin et al., 1991; Lockaby et al., 1998; Lockaby et al., 1999a; Lockaby et al., 1999b) and Morrow et al. (1990) suggested that variations in virulence may account for differences in the severity of the disease seen in broilers. There were no signs or lesions related to MS evident in the flock under study and the MS strain endemic on the farm seems to be of low virulence for broiler breeders and their offspring. Thus the lack of invasiveness of the MS may have been important for the success of its apparent eradication by the antibiotic treatments. The continued success of the treatment was probably related to the fact that at 22 weeks the flock was transferred to a clean farm, thus reducing the chances for reinfection from external sources. Several flocks emanating from the same rearing farm and housed untreated on other farms remained MS-positive by SPA until slaughter. MS was not isolated in this study, which is somewhat surprising although Fiorentin et al. (2002) reported that MS isolation is more likely between 27 and 28 weeks of age and our samples were collected at 23 weeks of age and then only after enrofloxacin treatment at 29 weeks. Persistant yeast contamination of the swabs may also have prejudiced isolation attempts. PCR proved more sensitive than isolation for detecting MS and was therefore useful for confirming the eradication of MS. HI tests were of low sensitivity and were negative when used on PCR positive birds. Antibodies were detected by SPA for many weeks after the PCR tests were negative but even these declined by the end of the study. These results suggest that surveillance for MS infection in antibiotic-treated flocks should not be based solely on serology but should be augmented by PCR. Insufficient samples were tested to confirm that MS was completely eradicated from the flock by antibiotic treatment and attention to biosecurity measures but all PCR reactions were negative by 47 weeks. The administration of a large dose of live IBV vaccine at 67 weeks did not provoke any recrudescence of MS but only five birds were challenged in this manner. Long term reliance on intensive antibiotic medication, particularly with fluoroquinolones, is no longer generally acceptable within the poultry industry but the targeted use of antibiotics in an eradication programme such as the one described here may reduce the need to medicate much larger numbers of birds in the next generation.
215
References Bradbury, J. (1998). Identification of mycoplasmas by immunofluorescence. In R. Miles & R. Nicholas (Eds.), Mycoplasma Protocols , 1st ed. (pp. 119 /126). Totowa: Humana Press. Cerda, R.O., Giacoboni, G.I., Xavier, J.A., Sansalone, P.L. & Landoni, M.F. (2002). In vitro antibiotic susceptibility of field isolates of Mycoplasma synoviae in Argentina. Avian Diseases , 46 , 215 /218. Chopra, I. & Roberts, M. (2001). Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews , 65 , 232 /260. Fiorentin, L., Balen, L. & Fialho, F.B. (1991). Patogenicidade de amostras de Mycoplasma synoviae isoladas no Brasil. Arquivo Brasileiro de Medicina Veterina´ria e Zootecnia , 43 , 405 /410. Fiorentin, L., Mores, M.A.Z., Trevisol, I.M., Antunes, S.C., da Costa, J.L.A., Soncini, R.A. & Vieira, N.D. (2002). Comportamento da infecc¸a˜o por Mycoplasma synoviae em matrizes de corte introduzidas em granja com histo´rico de positividade. Revista Brasileira de Cieˆncia Avı´cola , 4 (Suppl. 4), 122. Frey, M.L.L., Hanson, L.P. & Anderson, D.P. (1968). A medium for the isolation of avian mycoplasmas. American Journal of Veterinary Research , 19 , 2163 /2171. Hannan, P.C.T. (2000). Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasmas species. Veterinary Research , 31 , 273 /395. Hannan, P.C.T., Windsor, G.D., de Jong, A., Schmeer, N. & Stegemann, M. (1997). Comparative susceptibilities of various animal-pathogenic mycoplasmas to fluoroquinolones. Antimicrobial Agents and Chemotherapy , 41 , 2037 /2040. Jordan, F.T., Forrester, C.A., Ripley, P.H. & Burch, D.G. (1998). In vitro and in vivo comparisons of valnemulin, tiamulin, tylosin, enrofloxacin, and lincomycin/spectomycin against Mycoplasma gallisepticum . Avian Diseases , 42 , 738 /745. Kleven, S.H. (1998). Mycoplasmosis. In D.E. Swayne (Ed.), A Laboratory Manual for the Isolation and the Identification of Avian Pathogens , 4th ed. (pp. 74 /80). Kennett Square: The American Association of Avian Pathologists. Lauerman, L.H., Hoerr, F.J., Sharpton, A.R., Shah, S.M. & Van Santen, L. (1993). Development and application of a polymerase chain reaction assay for Mycoplasma synoviae . Avian Diseases , 37 , 829 /834. Lockaby, S.B., Hoerr, F.J., Lauerman, H.L. & Kleven, S.H. (1998). Pathogenicity of Mycoplasma synoviae in broiler chicken. Veterinary Pathology , 35 , 178 /190. Lockaby, S.B., Hoerr, F.J., Kleven, S.H. & Lauerman, H.L. (1999a). Pathogenicity of Mycoplasma synoviae in chicken embryos. Avian Diseases , 43 , 331 /337. Lockaby, S.B., Hoerr, F.J., Lauerman, H.L., Smith, B.F., Samoylov, A.M., Toivio-Kinnucan, M. & Kleven, S.H. (1999b). Factors associated with virulence of Mycoplasma synoviae . Avian Diseases , 43 , 251 /267. Luna, L.G. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology , 3rd ed.. New York: McGraw-Hill. Morrow, C.J., Bell, I.G., Walker, S.B., Markham, P.F., Thorp, B.H. & Whithear, K.G. (1990). Isolation of Mycoplasma synoviae from synovitis in chicken. Australian Veterinary Journal , 67 , 121 /124. Wellehan, J.F., Zens, M.S., Calsamiglia, M., Fusco, P.J., Amonsin, A. & Kapur, V. (2001). Diagnosis and treatment of conjunctivitis in house finches associated with mycoplasmosis in Minnesota. Journal of Wildlife Diseases , 37 , 245 /251.
RE´SUME´ Eradication de Mycoplasma synoviae chez des reproducteurs de type chair soumis a` des traitements antibiotiques intensifs pour controˆler Escherichia coli Un troupeau de reproducteurs de type chair indemne de Mycoplasma synoviae (MS) a e´te´ mis en place en pe´riode poulette dans une ferme ou` MS e´tait ende´mique. En neuf semaines, les re´sultats PCR ont re´ve´le´ que le troupeau s’e´tait infecte´. A l’aˆge de 22 semaines, le troupeau a e´te´
216 L. Fiorentin et al. transfe´re´ dans une ferme ou` la bande pre´ce´dente a e´te´ e´limine´e et les baˆtiments nettoye´s et de´sinfecte´s. Les oiseaux ont e´te´ soumis a` trois traitements de fluoroquinolone du fait qu’ils avaient pre´sente´ une pe´ritonite re´currente a` Escherichia coli et a` partir de l’aˆge de 32 semaines, ils ont rec¸u 600 ppm d’oxyte´tracycline hydrochloride en continu dans l’aliment. Les recherches de MS par PCR ont re´ve´le´ une diminution des oiseaux positifs apre`s l’aˆge de 34 semaines et MS a duˆ eˆtre e´radique´ a` en juger par les re´sultats constamment ne´gatifs en PCR. Nous en concluons que des traitements antibiotiques intensifs accompagne´s de mesures de biose´curite´ ade´quates peuvent e´liminer MS de reproducteurs de type chair infecte´s.
ZUSAMMENFASSUNG Eradikation von Mycoplasma synoviae bei Broilerelterntieren nach intensiver antibiotischer Therapie zur Beka¨mpfung von Escherichia coli Eine Mycoplasma synoviae (MS)-freie Mastelterntierherde wurde zur Brut und Aufzucht auf einer Farm mit endemischer MS untergebracht. Die Untersuchung mittels PCR ließ erkennen, dass sich die Herde innerhalb von neun Wochen infizierte. Mit 22 Wochen wurde die Herde in ein sauberes und desinfiziertes Stallgeba¨ude auf einer vorher leer gemachten Farm umgestallt. Wegen immer wieder auftretender durch Escherichia coli verursachten Peritonitis wurden bei den Tieren drei Behandlungen mit Fluorquinolon durchgefu¨hrt. Von der 32. Lebenswoche an erhielten sie 600 ppm Oxytetrazyklinhydrochlorid kontinuierlich im Futter. Die U¨berpru¨fung mit der PCR zeigte eine
Abnahme MS-positiver Tiere nach der 34. Lebenswoche. Aufgrund der nachfolgend dauerhaft negativen Ergebnisse in der PCR mag die MS-Infektion als eradikiert beurteilt werden. Wir schließen daraus, dass durch intensive antibiotische Behandlungen unterstu¨tzt durch ada¨quate Hygienemaßnahmen infizierte Mastelterntiere von MS befreit werden ko¨nnen.
RESUMEN Erradicacio´n de Mycoplasma synoviae en reproductores sujetos a un tratamiento antibio´tico intensivo para controlar Escherichia coli Un lote de reproductores libre de Mycoplasma synoviae (MS) fue alojado en una granja ende´mica de MS durante la crı´ a y recrı´ a. Mediante la te´cnica de PCR se detecto´ que el lote se infecto´ hasta las nueve semanas. A las 22 semanas el lote fue transferido a una caseta limpia y desinfectada en una granja que habı´ a sido despoblada. Las aves fueron tratadas tres veces con fluoroquinolonas debido a peritonitis recurrentes por Escherichia coli y a partir de las 32 semanas de edad recibieron 600 ppm de hidroclorido de oxitetraciclina continuamente en el pienso. La monitorizacio´n frente a MS mediante PCR demostro´ una disminucio´n de las aves positivas a MS tras las 34 semanas de edad y podrı´ a ser que el MS hubiera sido erradicado a juzgar por los resultados negativos obtenidos mediante PCR de forma consistente. Se concluye que un tratamiento antibio´tico intensivo junto con unas medidas de bioseguridad adecuadas puede eliminar el MS de reproductores infectados.
