Can fatty acids and oxytetracycline protect artificially raised larvae from developing European foulbrood?

Apidologie, Jul 2018

A quantitative assay for the transmission of European foulbrood (EFB) in artificially raised larvae was developed. This assay was used to determine the concentration of oxytetracycline (OTC) required to prevent larvae from developing EFB and whether 8 fatty acids (undecanoic, lauric [dodecanoic], myristic, myristoleic, ricinoleic, ricinelaidic, homo-y-linolenic and 13,16,19-docosatrienoic acids) which had previously been demonstrated to inhibit the growth of Melissococcus plutonius cultures, could protect larvae from developing EFB. The larval assay involved grafting individual larva (less than 24 hours old) into a single well in a micro-titre plate. Each larva was fed 10 μL of basic larval diet (BLD) containing 500 000 M. plutonius organisms. After 3 days the larvae were also fed 60 000 Paenibacillus alvei spores (a common secondary invader associated with EFB) in 10 μL BLD. The combination of these two organisms was required to reliably produce symptoms typical of that seen in field cases of EFB. Most larvae infected using this protocol died from EFB. To determine the efficacy of OTC, EFB infected larvae were fed 0, 1, 2.5, 5 10 or 20 μg/mL of OTC. Treatment with 1 μg/mL lowered the mortality rate from 93.75% to 69.5%. Treatments with 2.5 μg/mL to 10 μg/mL reduced the mortality rate further whereas treatment with 20 μg/mL reduced the rate to the same as the negative control. Larvae fed 20 or 200 μg/mL of each of the eight fatty acids were not protected from developing EFB.Die Europäische Faulbrut (EFB) ist im Vergleich zur Amerikanischen Faulbrut (AFB) eine relativ schlecht untersuchte Bakterienerkrankung. Anders als bei AFB, bei der lediglich ein Erreger in den Krankheitsprozess involviert ist, sind bei EFB eine ganze Reihe von sekundären Eindringlingen bekannt, von denen Paenibacillus alvei einer der häufigsten ist (Bailey, 1960). Da die Übertragung von EFB durch unkontrollierbare äußere Bedingungen sowie durch das Ausräumen von infizierten Larven durch Adultbienen behindert wird (Bailey, ), ist es schwierig, eine EFB-Erkrankung in Honigbienenvölker verlässlich durch künstliche Infektion zu erzeugen. Um die Bedeutung von Melissococcus plutonius und Paenibacillus alvei für die Entwicklung von EFB zu untersuchen und um die unterschiedlichen Behandlungsmethoden ohne Störung durch Adultbienen beurteilen zu können, entwickelten wir einen quantitativen Biotest für die Übertragung von EFB auf künstlich aufgezogene Larven.Dieser Larventest beinhaltete die Übertragung individueller Larven (jünger als 24 h) in eine Vertiefung einer Mikrotiter-Platte. Jede Larve wurde mit einer Basisdiät gefüttert, die 500 000 M. plutonius-Organismen enthielt. Nach 3 Tagen wurden die Larven zusätzlich mit 60 000 Sporen von P. alvei gefüttert, um den Verlauf von EFB besser zu simulieren. P. alvei ist ein verbreiteter sekundärer Eindringling im Zusammenhang mit EFB und eine Beimpfung mit M. plutonius allein ruft nicht die typischen klinischen Symptome einer EFB hervor.Oxytetrazyklin (OTC) ist derzeit das Antibiotikum der Wahl bei der Bekämpfung von EFB. Obwohl OTC seit Jahrzehnten zur Bekämpfung von EFB benutzt wird, ist nach wie vor unbekannt, welche Konzentration von OTC in einer Bienenlarve benötigt wird, um sie vor dem Ausbruch von EFB zu schützen. Um die Wirksamkeit von OTC zu bestimmen, wurden EFB-infizierte Larven mit 0, 1, 2,5, 5, 10 oder 20 μg/mL OTC gefüttert. Eine Behandlung mit lediglich 1 μg/mL reduzierte die Mortalität der Larven von 93,75 (Positivkontrolle) auf 69,53 %. Eine Konzentration von 20 μg/mL bot bereits einen Schutz, der vergleichbar mit der negativen Kontrolle war. Allerdings ist es unwahrscheinlich, dass eine solch hohe Konzentration benötigt wird, um EFB effektiv im Bienenvolk zu bekämpfen. Geringere Konzentrationen in Verbindung mit dem natürlichen Hygieneverhalten der Bienen würden vermutlich ausreichen, um klinische Symptome in Völkern mit EFB auszuschließen. Daher sollten Konzentrationen zwischen 2,5–10 μg/mL, für eine effektive Behandlung von EFB im Bienenvolk ausreichen.Falls Rückstandsprobleme vermieden werden müssen, sind alternative Methoden zur EFB-Kontrolle notwendig. Acht Fettsäuren (Undecan-, Laurin-, Myristin-, Myristolein-, Rizinol-, Ricinalaidin-, Linolen- und 13,16,19-Docosatrienoic-Säure), für die bereits eine Hemmung des Wachstums von M. plutonius- und P. larvae-Kulturen nachgewiesen wurden (Hornitzky, ), wurden im Larventest beurteilt. Keine der Fettsäuren bot einen Schutz der Larven gegenüber EFB. So war die Anzahl an toten Larven nach der Behandlung mit den Fettsäuren ähnlich hoch wie bei der unbehandelten Kontrolle. Die Tatsache, dass selbst hohe Dosen von 200 μg/mL an Fettsäuren keinen Schutz der Larven boten, während sehr kleine Konzentrationen von OTC (1 and 2,5 μg/mL) bereits die Larven schützte, untermauert die Unzweckmäßigkeit von Fettsäuren für die Bekämpfung.Diese Untersuchung zeigt klar, dass für den Test von Behandlungen sowohl in-vitro-Methoden (Laborkultur von M. plutonius) als auch in-vivo-Methoden (EFB-Larventest) verwendet werden sollten.

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Can fatty acids and oxytetracycline protect artificially raised larvae from developing European foulbrood?

Can fatty acids and oxytetracycline protect artificially raised larvae from developing European foulbrood?* Thomas Gi 0 1 Idris Bih 0 1 0 Private Mail Bag , 8, Camden, New South Wales, 2570 , Australia 1 Elizabeth Macarthur Agricultural Institute, New South Wales Department of Primary Industries 2 Manuscript editor: Peter Rosenkranz - A quantitative assay for the transmission of European foulbrood (EFB) in artificially raised larvae was developed. This assay was used to determine the concentration of oxytetracycline (OTC) required to prevent larvae from developing EFB and whether 8 fatty acids (undecanoic, lauric [dodecanoic], myristic, myristoleic, ricinoleic, ricinelaidic, homo-y-linolenic and 13,16,19-docosatrienoic acids) which had previously been demonstrated to inhibit the growth of Melissococcus plutonius cultures, could protect larvae from developing EFB. The larval assay involved grafting individual larva (less than 24 hours old) into a single well in a micro-titre plate. Each larva was fed 10 µL of basic larval diet (BLD) containing 500 000 M. plutonius organisms. After 3 days the larvae were also fed 60 000 Paenibacillus alvei spores (a common secondary invader associated with EFB) in 10 µL BLD. The combination of these two organisms was required to reliably produce symptoms typical of that seen in field cases of EFB. Most larvae infected using this protocol died from EFB. To determine the efficacy of OTC, EFB infected larvae were fed 0, 1, 2.5, 5 10 or 20 µg/mL of OTC. Treatment with 1 µg/mL lowered the mortality rate from 93.75% to 69.5%. Treatments with 2.5 µg/mL to 10 µg/mL reduced the mortality rate further whereas treatment with 20 µg/mL reduced the rate to the same as the negative control. Larvae fed 20 or 200 µg/mL of each of the eight fatty acids were not protected from developing EFB. 1. INTRODUCTION European foulbrood (EFB) is a relatively poorly understood bacterial disease when compared with American foulbrood (AFB), the other major bacterial disease of honey bees. Researchers have not commonly attempted to transmit EFB to honey bee colonies due to the unreliability in producing disease. This is probably because the transmission of EFB is hampered by uncontrollable environmental conditions and the ejection of infected larvae by adult bees from inoculated colonies (Bailey, 1960) . Inoculated colonies often re Techniques for transmitting EFB to colonies have been developed to instigate and confirm the cause of the disease (Tarr, 1936; Bailey, 1963; Bailey and Lochner, 1968) . However, only one reliable method for infecting colonies or laboratory raised larvae with M. plutonius has been published (McKee et al., 2004) . This method was successful in transmitting disease by feeding M. plutonius from EFB diseased material using EFB diseased larval extracts. However, the dose required to produce disease could only be estimated as the amount of infective material consumed by the larvae could not be accurately determined. EFB could not be transmitted by feeding M. plutonius cultures grown on an artificial medium, using the McKee et al. (2004) assay. Unlike AFB, in which only the causative agent is involved in the disease process, EFB is associated with a number of secondary invaders (Bailey, 1960) . One of the most common secondary invaders is Paenibacillus alvei. The role of P. alvei in the disease process is unknown. Oxytetracycline (OTC) is the antibiotic of choice for the treatment of EFB. However, there are still unanswered questions about EFB and its treatment with OTC that need further study. Although OTC has been used to treat EFB for decades, the concentration of OTC in honey bee larvae required to protect them from developing EFB is still unknown. This information would provide an indication as to how much OTC is required to protect honey bee larvae from EFB and how much OTC is required for colony treatment. Alternative methods for the control of EFB are needed if problems with residues are to be eliminated. In 1992 Shimanuki et al. (1992) demonstrated that chalkbrood mummies contained an antimicrobial substance which in laboratory tests inhibited the growth of P. larvae and M. plutonius. Feldlaufer et al. (1993) subsequently identified this substance to be the fatty acid linoleic acid. They also demonstrated that linoleic acid was active against P. larvae but did not test its effect on M. plutonius. Hornitzky (2003) tested 28 fatty acids for their activity against P. larvae and M. plutonius cultures. Fifteen of the fatty acids showed antibacterial activity against P. larvae and eight fatty acids showed activity against M. plutonius. As control agents fatty acids would be safe and environmentallysound (Feldlaufer et al., 1993) . Not only are these agents non-toxic to man but are actual foods and in the case of unsaturated fatty acids are essential for growth, development and health (Kabara, 1978) . The aims of this study were to develop a quantitative as (...truncated)


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Thomas Giersch, Idris Barchia, Michael Hornitzky. Can fatty acids and oxytetracycline protect artificially raised larvae from developing European foulbrood?, Apidologie, pp. 151-159, Volume 41, Issue 2, DOI: 10.1051/apido/2009066