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Avian Pathology (2003) 32, 213
/216 Apparent eradication of Mycoplasma synoviae inbroiler breeders subjected to intensive antibiotictreatment 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 1Brazilian Agricultural Research Corporation
/EMBRAPA, Embrapa Suı´nos e Aves, BR 153, km 110, VilaTamandua´, 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 MSendemic farm. PCR revealed that the flock became infected within nine weeks. At 22 weeks the flock wastransferred to a clean and disinfected house on a previously depopulated farm. The birds were then subjected tothree treatments with fluoroquinolones due to recurrent Escherichia coli peritonitis and from the 32 weeks ofage they received 600 ppm of oxytetracycline hydrochloride continuously in the feed. Monitoring by PCRshowed a decrease in MS positive birds after 34 weeks of age and MS may have been eradicated as judged byconsistent negative results in PCR. We conclude that intensive antibiotic treatments supported by adequatebiosecurity could clear MS from infected broiler breeders.
fortuitous finding that three in-feed treatments ofbroiler breeders with fluoroquinolones, coupled Mycoplasma synoviae (MS) infection is usually with treatment with oxytetracycline, which were asymptomatic in broiler breeders in Brazil but the aimed at controlling recurrent E. coli peritonitis, possibility that it might play an important role in also resulted in apparent eradication of MS from complex respiratory disease in the offspring has motivated breeding companies to consider eradica-tion. Broiler breeders are usually maintained underhigh biosecurity while broilers are often reared inareas where infectious bronchitis is endemic andEscherichia coli is a constant threat.
Material and Methods In the past, mycoplasma eradication pro- Source of broiler breeders grammes have been based on antibiotic or heattreatment of fertile eggs. Mycoplasmas are sensi- Broiler breeders were obtained from an MS- and Mycoplasmagallisepticum - (MG) free primary breeding stock farm in the south of tive to a number of different antibiotics including Brazil. The mycoplasma-free status had been confirmed by the tetracyclines (Cerda et al. , 2002; Chopra & Ro- Brazilian Ministry of Agriculture by repeated serological testing by berts, 2001) and fluoroquinolones (Cerda et al. , serum plate agglutination (SPA) and haemagglutination inhibition (HI) 2002; Hannan et al. , 1997; Jordan et al. , 1998; tests at 6 to 8 week intervals throughout the flock life, plus at least oneattempt to isolate MS from tracheal swabs. In addition we carried out Wellehan et al. , 2001). These are broad spectrum MS and MG polymerase chain reaction (PCR) on tracheal swabs from antibiotics and can be used to control a wide range birds of three ages (19, 33 and 66 weeks) and confirmed the negative of bacteria including E. coli . Here we report the *To whom correspondence should be addressed.
Tel.: /49 442-8555; Fax: /49 442-8559; E-mail: [email protected] 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 LtdDOI: 10.1080/0307945021000071641 L. Fiorentin et al.
Housing and flock history Tracheal swabs for PCR and culture and blood samples for RSA and HItests were also collected at 67 and 69 weeks from the 10 birds that The birds in this study were hatched at the grandparent company's were moved to isolators when the flock was slaughtered. These birds hatchery and brooded and reared on a high biosecurity farm with two were necropsied at 71 weeks and culture and PCR was attempted from 32 000 bird all-in all-out quarantine units. Asymptomatic MS infection, trachea, airsacs and hock joints. Internal organs and hock synovial usually detected by serology and occasionally confirmed by isolation membranes were examined for histological lesions (Luna, 1968).
from tracheal swabs, had occurred in every flock housed in recent years Minimal inhibitory concentrations (MICs) for ENR, NOR and OXT on the farm. At 22 weeks the birds were transferred to a farm that had were determined in vitro (Hannan, 2000) with the type strain WVU1853 been depopulated, cleaned and disinfected. They were housed in four and with two MS isolates (443 and 563) from the previous batch of houses and 12 000 of one house formed the population for the birds on the same brooding and rearing farm. PHO was not included as continued study. After 26 weeks the flock showed recurrent E. coli its mode of action is to inhibit peptidoglycan synthesis and therefore it peritonitis and received several in-feed antibiotic treatments. These has no activity against the cell wall-less mycoplasmas.
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 andhoused in isolators in two groups of five. One group was vaccinated by PCR indicated that the MS-free flock that was eye-drop with infectious bronchitis virus vaccine (Broilerbron-H120; placed on the MS endemic site was positive by 9 Schering-Plough Coopers) at 10 times the recommended dose in an weeks of age and 70% or more swabs were PCR attempt to exacerbate any MS infection. All 10 birds were necropsied at69 weeks.
positive up to 29 weeks (Table 1). After treatmentshad commenced these fell to 1 in 10 PCR positiveswabs by 34 weeks, thereafter there were no further Sampling and laboratory tests positive PCR reactions. SPA reactions were firstseen at 29 weeks and persisted until 59 weeks. By Ten tracheal swabs for PCR and 10 more for mycoplasma isolationwere collected at 9, 15, 23, 29, 34, 47, 54 and 59 weeks of age together the time the flock was slaughtered at 67 weeks with 20 blood samples. PCRs were carried out on individual swabs by there were no positive SPA reactors and the 10 the method of Lauerman et al. (1993). Mycoplasma isolation was hens that were retained, including the five that attempted for each swab in 3 ml of broth (Frey et al. , 1968) received live I BV vaccine, remained PCR and SPA supplemented with 0.1 g/l nicotinamide adenine dinucleotide and 0.1 negative. No positive HIreactions were recorded g/l cysteine hydrochloride. Broths showing pH change or turbidity wereplated onto agar (Frey broth with 0.75% agarose). All other broths during the entire study nor were signs or lesions were plated out weekly for three weeks. Mycoplasma isolates were related to MS seen in either the flock prior to identified by immunofluorescence and filter-cloned three times (Brad- slaughter or in the 10 retained birds. No micro- bury, 1998).
scopic lesions of airsacculitis or synovitis were seen SPA tests were performed on the farm on fresh sera using in the tissues taken from these 10 birds.
commercially prepared antigen (Intervet B.V., Boxmeer, the Nether-lands) and the HItest (Kleven and Mycoplasmosis, 1998) was carried MIC breakpoints were 2 mg/ml for OXT, 0.125 out on the serum collected at 29 weeks.
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 Isolation (of 10) MS-free flock housed on MS endemic farm Flock moved to a clean farm E. coli peritonitis diagnosed; 35 mg/kg phosphomycin in feed for 5 d 10 mg/kg enrofloxacin in feed for 5 d 600 ppm oxytetracycline started in feed (continuous) 10/ mg/kg enrofloxacin for 5 d 10 mg/kg norfloxacin for 5 d Flock slaughtered; 10 hens moved to isolators; 5/10 given 10 / IBV0 Remaining 10 birds necropsied; no lesions seen aout of 20 birds.
bout of 10 birds.
Mycoplasma synoviae eradication breakpoints for the type strain were 2 mg/ml for OXT and ENR and 1 mg/ml for NOR. All thesewere considered to be in the sensitive range.
Bradbury, J. (1998). Identification of mycoplasmas by immunofluores- cence. In R. Miles & R. Nicholas (Eds.), Mycoplasma Protocols , 1sted. (pp. 119
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Cerda, R.O., Giacoboni, G.I., Xavier, J.A., Sansalone, P.L. & Landoni, M.F. (2002). In vitro antibiotic susceptibility of field isolates ofMycoplasma synoviae in Argentina. Avian Diseases , 46 , 215
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It has been reported that MS strains can differ in Chopra, I. & Roberts, M. (2001). Tetracycline antibiotics: mode of virulence (Fiorentin et al., 1991; Lockaby et al., action, applications, molecular biology, and epidemiology of bacter- 1998; Lockaby et al. , 1999a; Lockaby et al. , 1999b) ial resistance. Microbiology and Molecular Biology Reviews , 65 ,232
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and Morrow et al. (1990) suggested that variations Fiorentin, L., Balen, L. & Fialho, F.B. (1991). Patogenicidade de in virulence may account for differences in the amostras de Mycoplasma synoviae isoladas no Brasil. Arquivo severity of the disease seen in broilers. There were Brasileiro de Medicina Veterina´ria e Zootecnia , 43 , 405
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no signs or lesions related to MS evident in the Fiorentin, L., Mores, M.A.Z., Trevisol, I.M., Antunes, S.C., da Costa, flock under study and the MS strain endemic on J.L.A., Soncini, R.A. & Vieira, N.D. (2002). Comportamento dainfecc¸a˜o por Mycoplasma synoviae em matrizes de corte introduzidas the farm seems to be of low virulence for broiler em granja com histo´rico de positividade. Revista Brasileira de Cieˆncia breeders and their offspring. Thus the lack of Avı´cola , 4 (Suppl. 4), 122.
invasiveness of the MS may have been important Frey, M.L.L., Hanson, L.P. & Anderson, D.P. (1968). A medium for for the success of its apparent eradication by the the isolation of avian mycoplasmas. American Journal of Veterinary antibiotic treatments. The continued success of the Research , 19 , 2163
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Hannan, P.C.T. (2000). Guidelines and recommendations for anti- treatment was probably related to the fact that at microbial minimum inhibitory concentration (MIC) testing against 22 weeks the flock was transferred to a clean farm, veterinary mycoplasmas species. Veterinary Research , 31 , 273
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thus reducing the chances for reinfection from Hannan, P.C.T., Windsor, G.D., de Jong, A., Schmeer, N. & external sources. Several flocks emanating from the Stegemann, M. (1997). Comparative susceptibilities of various same rearing farm and housed untreated on other animal-pathogenic mycoplasmas to fluoroquinolones. AntimicrobialAgents and Chemotherapy , 41 , 2037
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farms remained MS-positive by SPA until slaugh- Jordan, F.T., Forrester, C.A., Ripley, P.H. & Burch, D.G. (1998). In vitro and in vivo comparisons of valnemulin, tiamulin, tylosin, MS was not isolated in this study, which is enrofloxacin, and lincomycin/spectomycin against Mycoplasma gal- somewhat surprising although Fiorentin et al.
lisepticum . Avian Diseases , 42 , 738
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(2002) reported that MS isolation is more likely Kleven, S.H. (1998). Mycoplasmosis. In D.E. Swayne (Ed.), A Laboratory Manual for the Isolation and the Identification of Avian between 27 and 28 weeks of age and our samples Pathogens , 4th ed. (pp. 74
/80). Kennett Square: The American were collected at 23 weeks of age and then only Association of Avian Pathologists.
after enrofloxacin treatment at 29 weeks. Persistant Lauerman, L.H., Hoerr, F.J., Sharpton, A.R., Shah, S.M. & Van yeast contamination of the swabs may also have Santen, L. (1993). Development and application of a polymerase prejudiced isolation attempts.
chain reaction assay for Mycoplasma synoviae . Avian Diseases , 37 ,829
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PCR proved more sensitive than isolation for Lockaby, S.B., Hoerr, F.J., Lauerman, H.L. & Kleven, S.H. (1998).
detecting MS and was therefore useful for con- Pathogenicity of Mycoplasma synoviae in broiler chicken. Veterinary firming the eradication of MS. HItests were of low Pathology , 35 , 178
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sensitivity and were negative when used on PCR Lockaby, S.B., Hoerr, F.J., Kleven, S.H. & Lauerman, H.L. (1999a).
positive birds. Antibodies were detected by SPA Pathogenicity of Mycoplasma synoviae in chicken embryos. AvianDiseases , 43 , 331
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for many weeks after the PCR tests were negative Lockaby, S.B., Hoerr, F.J., Lauerman, H.L., Smith, B.F., Samoylov, but even these declined by the end of the study.
A.M., Toivio-Kinnucan, M. & Kleven, S.H. (1999b). Factors These results suggest that surveillance for MS associated with virulence of Mycoplasma synoviae . Avian Diseases , infection in antibiotic-treated flocks should not 43 , 251
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be based solely on serology but should be aug- Luna, L.G. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology , 3rd ed. New York: McGraw-Hill.
mented by PCR.
Morrow, C.J., Bell, I.G., Walker, S.B., Markham, P.F., Thorp, B.H. & Insufficient samples were tested to confirm that Whithear, K.G. (1990). Isolation of Mycoplasma synoviae from MS was completely eradicated from the flock by synovitis in chicken. Australian Veterinary Journal , 67 , 121
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antibiotic treatment and attention to biosecurity Wellehan, J.F., Zens, M.S., Calsamiglia, M., Fusco, P.J., Amonsin, A.
measures but all PCR reactions were negative by & Kapur, V. (2001). Diagnosis and treatment of conjunctivitis inhouse finches associated with mycoplasmosis in Minnesota. Journal 47 weeks. The administration of a large dose of live of Wildlife Diseases , 37 , 245
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IBV vaccine at 67 weeks did not provoke anyrecrudescence of MS but only five birds werechallenged in this manner.
Long term reliance on intensive antibiotic med- Eradication de Mycoplasma synoviae chez des reproducteurs de type ication, particularly with fluoroquinolones, is no chair soumis a des traitements antibiotiques intensifs pour controˆler longer generally acceptable within the poultry industry but the targeted use of antibiotics in an Un troupeau de reproducteurs de type chair indemne de Mycoplasma eradication programme such as the one described synoviae (MS) a e´te´ mis en place en pe´riode poulette dans une ferme ou here may reduce the need to medicate much larger MS e´tait ende´mique. En neuf semaines, les re´sultats PCR ont re´ve´le´ que numbers of birds in the next generation.
le troupeau s'e´tait infecte´. A l'aˆge de 22 semaines, le troupeau a e´te´ L. Fiorentin et al.
transfe´re´ dans une ferme ou la bande pre´ce´dente a e´te´ e´limine´e et les Abnahme MS-positiver Tiere nach der 34. Lebenswoche. Aufgrund baˆtiments nettoye´s et de´sinfecte´s. Les oiseaux ont e´te´ soumis a trois der nachfolgend dauerhaft negativen Ergebnisse in der PCR mag die traitements de fluoroquinolone du fait qu'ils avaient pre´sente´ une MS-Infektion als eradikiert beurteilt werden. Wir schließen daraus, pe´ritonite re´currente a Escherichia coli et a partir de l'aˆge de dass durch intensive antibiotische Behandlungen unterstu¨tzt durch 32 semaines, ils ont rec¸u 600 ppm d'oxyte´tracycline hydrochloride en ada¨quate Hygienemaßnahmen infizierte Mastelterntiere von MS befreit continu dans l'aliment. Les recherches de MS par PCR ont re´ve´le´ une werden ko¨nnen.
diminution des oiseaux positifs apre s l'aˆge de 34 semaines et MS a duˆeˆtre e´radique´ a en juger par les re´sultats constamment ne´gatifs en PCR.
Nous en concluons que des traitements antibiotiques intensifs accom- pagne´s de mesures de biose´curite´ ade´quates peuvent e´liminer MS de Erradicacio´n de Mycoplasma synoviae en reproductores sujetos a un reproducteurs de type chair infecte´s.
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 Eradikation von Mycoplasma synoviae bei Broilerelterntieren nach nueve semanas. A las 22 semanas el lote fue transferido a una caseta intensiver antibiotischer Therapie zur Beka¨mpfung von Escherichia coli limpia y desinfectada en una granja que habı´a sido despoblada. Las Eine Mycoplasma synoviae (MS)-freie Mastelterntierherde wurde zur aves fueron tratadas tres veces con fluoroquinolonas debido a Brut und Aufzucht auf einer Farm mit endemischer MS untergebracht.
peritonitis recurrentes por Escherichia coli y a partir de las 32 semanas Die Untersuchung mittels PCR ließ erkennen, dass sich die Herde de edad recibieron 600 ppm de hidroclorido de oxitetraciclina innerhalb von neun Wochen infizierte. Mit 22 Wochen wurde die Herde continuamente en el pienso. La monitorizacio´n frente a MS mediante in ein sauberes und desinfiziertes Stallgeba¨ude auf einer vorher leer PCR demostro´ una disminucio´n de las aves positivas a MS tras las 34 gemachten Farm umgestallt. Wegen immer wieder auftretender durch semanas de edad y podrı´a ser que el MS hubiera sido erradicado a Escherichia coli verursachten Peritonitis wurden bei den Tieren drei juzgar por los resultados negativos obtenidos mediante PCR de forma Behandlungen mit Fluorquinolon durchgefu¨hrt. Von der 32. Lebens- consistente. Se concluye que un tratamiento antibio´tico intensivo junto woche an erhielten sie 600 ppm Oxytetrazyklinhydrochlorid konti- con unas medidas de bioseguridad adecuadas puede eliminar el MS de nuierlich im Futter. Die U¨berpru¨fung mit der PCR zeigte eine

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