Isolation and antimicrobial susceptibility of Brachy -spira species from feces of layer chickens in Germany

Summary

Objective: Anaerobic spirochetes of the genus Brachyspira are important pathogens causing swine dysentery (Brachyspira [B.] hyodysenteriae) and porcine intestinal spirochetosis (B. pilosicoli, PIS). In addition, avian intestinal spirochetosis (AIS) is caused by B. pilosicoli, B. intermedia and B. alvinipulli. Despite the economic impact of AIS, the disease has not received appropriate attention in Germany. This study was aimed at identifying Brachyspira spp. in Germany and determining their antimicrobial susceptibility. Material and methods: From 2009 to 2013, a total of 71 fecal swabs were obtained from clinically healthy layer hens from eight different commercial flocks. Brachyspira spp. culture was performed in trypticase soybean agar added with 5% sheep blood. Species determination was conducted by PCRs targeting the NADH-gen and the 16S rDNA or by nox-gene sequencing. Antimicrobial susceptibility to macrolides, lincosamides and pleuromutilins was tested by a microdilution assay. Results: Brachyspira spp. were isolated from 40 (56.3%) swabs distributed over all eight flocks. In 26 cases, the following species were determined by PCR: B. pilosicoli (n = 16), B. intermedia (2), B. innocens (3), B. murdochii (1), mixtures of B. pilosicoli/B. intermedia (2), B. innocens/B. intermedia (1), B. innocens/B. murdochii (1). Remaining isolates were characterized by noxgene sequencing as B. “pulli” (n = 9), B. alvinipulli (3), B. intermedia (1) and as not identifiable (1). Antimicrobial susceptibility testing of 37 isolates revealed minimal inhibitory concentrations 90 (MIC₉₀) of > 128 mg/l (tylosin), 64 mg/l (lincomycin), 8 mg/l (tiamulin) and 4 mg/l (valnemulin), respectively. Comparing to breakpoints applied to pigs, these values lie within the range of resistance. Conclusion The demonstration of different Brachyspira spp., particularly B. pilosicoli, intermedia and alvinipulli in commercial layers, indicates the need of further research to assess their potential role in causing AIS in German poultry flocks. The increased antimicrobial resistance of Brachyspira spp. isolates to tylosin and pleuromutilins is likely associated with extensive use of these drugs in poultry medicine.

Zusammenfassung

Gegenstand und Ziel Die zum Genus Brachyspira zählenden obligat anaeroben Spirochäten verursachen die Schweinedysenterie (Erreger: Brachyspira [B.] hyodysenteriae) sowie die porzine (PIS) und aviäre (AIS) intestinale Spirochaetose (B. pilosicoli bzw. im letzteren Fall auch B. intermedia und B. alvinipulli). Trotz potenzieller Bedeutung der AIS erhält diese Krankheit in Deutschland wenig Aufmerksamkeit. Ziel der Studie war der Nachweis von Brachyspira spp. beim Geflügel und Prüfung deren antimikrobieller Resistenz. Material und Methoden: Zur kulturellen Untersu chung dienten 71 zwischen 2009 und 2013 entnommene Kottupferproben in Amies Transportmedium von klinisch unauffälligen Legehennen aus acht Herden. Die Isolate wurden mittels PCRs, die auf Abschnitte im NADH-Gen und die 16S rDNA zielten, oder mittels nox-Gen-Sequenzierung identifiziert und anschließend im Mikrodilutionstest zur Ermittlung der minimalen Hemmkonzentration (MHK) von Makroliden, Lincosamiden und Pleuromutilinen untersucht. Ergebnisse Von 40 (56,3%) Kottupfern wur den schwach hämolysierende Brachyspira spp. kultiviert. Mittels PCR waren davon jedoch lediglich 26 Isolate bis zur Speziesebene identifizierbar: B. pilosicoli (n = 16), B. intermedia (2), B. innocens (3), B. murdochii (1), B. pilosicoli/B. intermedia (2), B. innocens/B. intermedia (1), B. innocens/B. murdochii (1). Verbliebene Isolate wurden mittels nox-Gen-Sequenzierung als B. “pulli” (n = 9), B. alvinipulli (3), B. intermedia (1) sowie als nicht identifizierbar charakterisiert (1). Die Antibiotikaempfindlichkeits prüfung von 37 Isolaten ergab MHK₉₀-Werte von > 128 mg/l (Tyosin), 64 mg/l (Lincomycin), 8 mg/l (Tiamulin) bzw. 4 mg/l (Valnemulin). Bezogen auf die für das Schwein ermittelten Grenzwerte liegen die Werte im Bereich für Resistenz. Schlussfolgerung und klinische Relevanz: Infolge des Nachweises ver schiedener schwach hämoly-sierender Brachyspira spp., inklusive der mit AIS ätiologisch assoziierten Spezies B. pilosicoli, intermedia und alvinipulli, erscheinen Untersuchungen zu deren klinischer Bedeutung bei Legehennen in Deutschland angezeigt. Die geringe Antibiotikaempfindlichkeit der Isolate ist sehr wahrscheinlich auf die weit verbreitete Anwendung von Tylosin und Pleuromutilinen beim Geflügel zurückzuführen.

Keywords

Poultry - spirochetes - occurrence - antimicrobial susceptibility

SchlüsselWörter

Geflügel - Spirochäten - Vorkommen - Antibiotikaempfindlichkeit

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