Monitoring of Small Hive Beetle (Aethina Tumida Murray) in Calabria (Italy) from 2014 to 2016: Practical Identification Methods

Abstract The Small Hive Beetle (SHB), Aethina tumida, is an invasive pest of honey bee colonies that causes significant damage to the beekeeping sector. SHB was detected in southern Italy (EU) in 2014 and despite adopted eradication measures, is still present there. After three years of observations of SHB in Calabria (2014-2016), we provide here some practical tips for improving control measures. A new time-saving colony examination method, including the use of an internal divider reduced the time needed for hive inspections by 31.86 % on average. Prioritizating the inspection of pollen and honey combs rather than brood combs is advised. Sentinel apiaries with no more than five colonies without supers are suggested for each beekeeping location in order to attract and to monitor the early appearance of SHB. The use of these methods will enable early detection and prompt control measures application before this destructive pest can spread in the region

Oregano Essential Oil and Purple Garlic Powder Effects on Intestinal Health, Microbiota Indicators and Antimicrobial Resistance as Feed Additives in Weaning Piglets

Finding alternatives to zinc oxide is a pressing issue for the pig production sector. We studied the impact of the bioactive components degradation of oregano essential oil (OEO) and purple garlic powder (PGP) during storage in silos, their effect on the morphometry of the jejunum and ileum and the cecal microbiota as intestinal health indicators in piglets during the post-weaning period. We also monitored antimicrobial resistance in the commensal indicator E. coli. Histological parameters and intestinal microbiota were measured in 140 piglets weaned at 21 days of age. Seven dietary treatments were used: a negative control group (basal diet), a positive control group with ZnO (3000 mg/kg of food), two groups with OEO at 0.4% and 1.2%, respectively, two groups with PGP 0.4% and 2%, respectively, and a group with OEO with 1.2% combined with PGP with 2%. Each group of piglets received the treatment for seven weeks, from weaning, before samples were taken. Antibiotic resistance profiles were measured in 81 E. coli strains. On this occasion, only the control groups, ZnO, OEO 1.2%, PGP 2% and OEO 1.2% + PGP 2% were used, and the samples were obtained from the cecal content. A progressive loss of the bioactive components of OEO and PGP was observed during the 34 days of storage (p < 0.05). PGP 2%, OEO 1.2% and their combination showed results similar to ZnO (p > 0.05), or superior in the study of intestinal morphometry and the values of E. coli and Lactobacillus. All categories showed high levels of resistance. Only the strains isolated from the OEO 1.2% group did not show resistance to colistin and presented the lowest resistance values. In general, high doses of the additives studied showed the best results, obtaining levels like or higher than those offered by ZnO

Monitorización de resistencias a los antimicrobianos en producción animal y empleo de aditivos vegetales como alternativas a los antibióticos y al óxido de zinc

La presente tesis doctoral se ha realizado en formato de compendio de publicaciones y está compuesta por tres artículos publicados en revistas internacionales (Q1). Todos los artículos constituyen una unidad científica, en el marco de la investigación sobre resistencia a los antimicrobianos en producción animal y al desarrollo de productos alternativos a los antimicrobianos y al óxido de zinc (ZnO). Dos de las publicaciones se centraron en estudiar la presencia y caracterización de la resistencia a los antimicrobianos en bacterias comensales, presentes en granjas de aves de puesta en España. Esto se debió a la escasez de datos actuales en gallinas ponedoras, en contraste con otros sectores más estudiados y monitorizados a nivel español y europeo, como la producción avícola de carne. Durante la realización de estos trabajos, se estudió la resistencia a los antibióticos según las categorías establecidas por la Agencia Europea del Medicamento (EMA), prestando especial atención a la resistencia a antibióticos de especial importancia para medicina humana, con un enfoque de “Una Salud”. El primer estudio trató sobre la resistencia a antibióticos de categorías C y D en Campylobacter spp., Escherichia coli y Enterococcus spp. comensales en granjas de gallinas ponedoras españolas, que se determinó mediante la Concentración Mínima Inhibitoria (CMI). Se encontraron altos valores de resistencia a diversos antibióticos, incluyendo algunos de importancia para el tratamiento de zoonosis. Se encontraron valores de resistencia particularmente altos para la tetraciclina, familia tetraciclinas, de la categoría D, y para la eritromicina, familia macrólidos, de la categoría C. El segundo estudio, se centró en la resistencia de Campylobacter jejuni, Escherichia coli y Enterococcus faecalis a antimicrobianos de las categorías A y B mediante determinación de la CMI. Se encontraron niveles de resistencia altos a medicamentos de importancia para medicina humana, y los niveles detectados fueron diferentes de los datos disponibles para aves de corral en España y en la UE. Hubo valores especialmente preocupantes para ciprofloxacino y ácido nalidíxico, familia quinolonas, de la categoría B, y para la tigeciclina, familia glicilciclinas, de la categoría A. De los resultados de estos dos primeros trabajos se puede concluir que las granjas de gallinas ponedoras en España presentan niveles altos de resistencias a todas las categorías de antibióticos (A, B, C y D), incluyendo los usados en medicina humana. Esto supone un riesgo para la salud pública desde el punto de vista de “Una Salud”. Además, las diferencias encontradas entre la información previamente disponible y estos resultados, hacen evidente que son necesarios métodos de muestreo y monitorización más detallados para el sector de las gallinas ponedoras. Estas investigaciones suponen un avance hacia la correcta caracterización de las resistencias a los antimicrobianos en producción animal, que es esencial para permitir el diseño e implementación de medidas apropiadas, con el objetivo de prevenir el aumento de las resistencias y preservar la eficacia de los antimicrobianos. En producción animal, el desarrollo de métodos y productos alternativos a los antibióticos es un paso esencial para prevenir y reducir la presencia de resistencias a los antimicrobianos. La producción porcina intensiva es uno de los principales sectores consumidores de antibióticos y productos con similares características como el ZnO. El destete de los lechones es uno de los puntos más críticos del ciclo productivo, y para prevenir problemas digestivos se suele usar el ZnO, que provoca graves problemas de contaminación ambiental y contribuye al aumento de las resistencias a los antimicrobianos. En la tercera publicación de esta tesis, se estudiaron los efectos de suplementar moltura de ajo morado (A. sativum) y aceite esencial de orégano (O. vulgare L.) microencapsulado, sobre biomarcadores del estado oxidativo, estrés e inflamación, así como sobre la ganancia media diaria y el índice de conversión, en comparación con el ZnO y la dieta control, en lechones destetados. Las dosis de aceite esencial de orégano microencapsulado y moltura de ajo al 0,4% no provocaron inflamación, estrés o cambios negativos en los biomarcadores oxidativos en los lechones, y dieron un mejor rendimiento productivo que las dietas control y ZnO. Estos resultados reflejan que los productos derivados del ajo y del orégano como aditivos en el pienso, gracias a sus propiedades beneficiosas, pueden contribuir a reducir la necesidad de usar antibióticos y ZnO en producción animal, permitiendo mantener los animales en un buen estado sanitario y productivo. Esto ayuda a reducir el uso de antimicrobianos y ZnO, contribuyendo a la sostenibilidad de la producción animal y a la protección de la salud animal, humana y medioambiental dentro de un contexto de “Una Salud”.This PhD thesis has been written as a compendium of publications and is composed of three papers published in international journals (Q1). All the publications form a scientific unit within the framework of research on antimicrobial resistance in animal production and the development of alternative products to antimicrobials and zinc oxide (ZnO). Two of the publications studied the presence and characterization of antimicrobial resistance in commensal bacteria in laying hens farms in Spain. This studies were carried out due to the scarcity of current data on laying hens, in contrast to other sectors more studied and monitored at the Spanish and European level, such as broiler production. In these studies, resistance to antibiotics was studied according to the categories established by the European Medicines Agency (EMA), paying special attention to antibiotics of special importance for human medicine, with a "One Health” approach. The first study dealt with resistance to category C and D antibiotics in commensal Campylobacter spp., Escherichia coli and Enterococcus spp. in Spanish laying hen farms, which was determined by the Minimum Inhibitory Concentration (MIC). High values of resistance to various antibiotics were found, including some important for the treatment of zoonoses. Particularly high resistance values were found for tetracycline, tetracyclines family, from category D, and for erythromycin, macrolides family, from category C. The second study focused on the resistance of Campylobacter jejuni, Escherichia coli and Enterococcus faecalis to antimicrobial categories A and B by determining the MIC. High levels of resistance were found to important drugs for human medicine, and the results were different from the data available for poultry in Spain and in the EU. There were values particularly concerning for ciprofloxacin and nalidixic acid, quinolone family, from category B, and for tigecycline, glycylcyclines family, from category A. From the results of these two studies, it can be concluded that laying hen farms in Spain have high levels of resistance to all categories of antimicrobials (A, B, C and D), including those used in human medicine. This poses a risk to public health from a “One Health” perspective. Furthermore, the differences found between the information previously available and these results, evidence that more detailed sampling and monitoring methods are necessary for the laying hen sector. These investigations represent a step towards the correct characterization of antimicrobial resistance in animal production, which is essential to make possible the design and implementation of appropriate measures, with the aim of preventing the increase in resistance and preserving the efficacy of antimicrobials. The development of methods and products alternative to antibiotics is an essential step to prevent and reduce the presence of antimicrobial resistance in animal production. Intensive pig production is one of the main consumer sectors for antibiotics and products with similar characteristics such as ZnO. Weaning is one of the most critical points in the pig production cycle, and ZnO is often used to prevent digestive problems, which causes serious environmental pollution and contributes to the increase of resistance to antimicrobials. Due to these negative consequences, ZnO will be banned in the EU from June 2022. In the third publication of this thesis, the effects of supplementing purple garlic (A. sativum) powder and micro-encapsulated oregano (O. vulgare L.) essential oil were studied on biomarkers of oxidative status, stress and inflammation, as well as on the average daily gain and the feed conversion index, compared to ZnO and the control diet, in weaned piglets. The doses of micro-encapsulated oregano essential oil and 0.4% garlic powder did not cause inflammation, stress or negative changes in oxidative biomarkers in piglets, and gave a better productive performance than the control and ZnO diets. These results show that the products derived from garlic and oregano as feed additives, thanks to their beneficial properties, can allow to reduce the need of using antibiotics and ZnO in animal production, since they contribute to keep the animals in good sanitary and productive conditions. This helps to reduce the use of antimicrobials and ZnO, contributing to the sustainability of animal production and the protection of animal, human and environmental health within a “One Health” context

Monitoring of Small Hive Beetle (Aethina Tumida Murray) in Calabria (Italy) from 2014 to 2016: Practical Identification Methods

The Small Hive Beetle (SHB), Aethina tumida, is an invasive pest of honey bee colonies that causes significant damage to the beekeeping sector. SHB was detected in southern Italy (EU) in 2014 and despite adopted eradication measures, is still present there. After three years of observations of SHB in Calabria (2014-2016), we provide here some practical tips for improving control measures. A new time-saving colony examination method, including the use of an internal divider reduced the time needed for hive inspections by 31.86 % on average. Prioritizating the inspection of pollen and honey combs rather than brood combs is advised. Sentinel apiaries with no more than five colonies without supers are suggested for each beekeeping location in order to attract and to monitor the early appearance of SHB. The use of these methods will enable early detection and prompt control measures application before this destructive pest can spread in the region

Nosemosis Prevention and Control

Nosemosis is a serious microsporidian disease of adult European honey bees caused by the spore-forming unicellular fungi Nosema apis and Nosema ceranae. In this paper we describe the currently known techniques for nosemosis prevention and control including Good Beekeeping Practices (GBPs) and biosecurity measures (BMBs). Topics such as queen renewal, nosema-resistant bees and hygienic and control methods are described. Strong efforts are currently provided to find more a sustainable solution than the use of antibiotics. So far, it seems that the best way to approach nosemosis is given by an “integrated pest management strategy”, which foresees the contemporary application of different, specific GBPs and BMBs

Antimicrobial Resistance of Campylobacter jejuni, Escherichia coli and Enterococcus faecalis Commensal Isolates from Laying Hen Farms in Spain

Antimicrobial resistance (AMR) is a global threat for human and animal health. Few studies have been carried out in laying hens. We evaluated the antimicrobial susceptibility of commensal Campylobacter jejuni, Escherichia coli, and Enterococcus faecalis isolates in Spanish laying hens in 2018. The Minimum Inhibitory Concentration (MIC) was used to identify any AMR of the studied isolates by means of a broth microdilution method. C. jejuni was highly resistant to the B category antimicrobials, and 52% of the isolates were susceptible to all the antimicrobials tested. E. coli showed medium and high percentages of resistance to the B and A antibiotic categories, respectively, and 33.33% of the isolates were susceptible to all antimicrobials. The E. faecalis resistance to A category antimicrobials was variable, and 4.62% of the isolates were susceptible to all antimicrobials. In our work, novel data on AMR in laying hen commensal isolates in Spain is provided, and the AMR levels differ from those reported for poultry in the EU. A high resistance to key drugs for human medicine was found, representing a public health risk

Effects of Dietary Supplementation of Garlic and Oregano Essential Oil on Biomarkers of Oxidative Status, Stress and Inflammation in Postweaning Piglets

The effects of two different concentrations of micro capsuled oregano essential oil (OEO) and purple garlic powder on biomarkers of oxidative status, stress, and inflammation, as well as on average daily gain (ADG) and feed conversion ratio (FCR), were evaluated in piglets during the postweaning period. The trial was carried out with 300 crossbred pigs of 21 days of age fed with different concentrations of OEO and purple garlic powder and ZnO. Saliva and serum samples were taken to evaluate a panel of biomarkers of oxidative status, stress, and inflammation. OEO and garlic powder at 0.4% did not produce significant changes in C-reactive protein (CRP) and cortisol and yielded higher levels of the antioxidant biomarker CUPRAC in serum than higher doses (p < 0.01); they yielded a better ADG than the control and ZnO diets. OEO and garlic powder at higher concentrations than 0.4% showed higher concentrations of CRP (p < 0.05). Overall, doses of OEO and garlic powder at 0.4% did not lead to inflammation, stress, or negative changes in oxidative biomarkers in piglets during the postweaning period and gave better productive performance than the control and ZnO diets. High doses of OEO and garlic powder were ineffective and could negatively affect the animals. Therefore, our results highlight the importance of the dose used when OEO or garlic are supplemented to piglets

How to slow the global spread of small hive beetles, Aethina tumida

Small hive beetles (SHBs) are parasites of social bee colonies endemic to sub-Saharan Africa and have become a widespread invasive species. In the new ranges, SHBs can cause damage to apiculture and wild bees. Although the further spread seems inevitable, eradication of new introductions and containment of established ones are nevertheless urgently required to slow down the invasion speed until better mitigation options are available. However, at present there is no adequate action plan at hand. Here, we propose to take advantage of SHB invasion history and biology to enrol a feasible plan involving all stakeholders. Raising awareness, education and motivation of stakeholders (incl. adequate and timely compensation of beekeepers) is essential for success. Moreover, sentinel apiaries are recommended in areas at risk, because early detection is crucial for the success of eradication efforts. Given that introductions are detected early, SHB eradication is recommended, incl. destruction of all infested apiaries, installation of sentinel colonies to lure escaped SHBs and a ban on migratory beekeeping. If wild perennial social bee colonies are infested, eradication programs are condemned to fail and a strategic switch to a containment strategy is recommended. Containment includes adequate integrated pest management and a strict ban on migratory beekeeping. Despite considerable gaps in our knowledge of SHBs, the proposed action plan will help stakeholders to slow down the global spread of SHBs