**Author details**

*larvae* [123, 124, 130, 134], and the causative agent of American foulbrood, among others. This effectiveness is generally associated with the production of acid, bacteriocins [130], and other

Traditional methods are often still used for monitoring the microbiological quality of honey used for human consumption, even as the rates established by the laws use these methods. Potato dextrose agar and yeast extract glucose chloramphenicol agar are media normally used for aerobic count and the total fungi (yeasts and molds), while Violet Red Bile and MacConkey medium agars are normally used for counting coliforms, which can also be done by the most probable number technique [119, 141, 142]. These media have recently been used to monitor the efficiency of a new filter-based method based in reducing the microbial burden and to improve the microbiological quality of honey [143]. Potato glucose agar in Brazil was also used for monitoring the honey contamination by yeast and fungi [144]. Standard plate count agar is used for monitoring of mesophilic bacteria, such as that was done in honey

The honey is an important route of contamination of honeybees, spreading many microorganisms, particularly pathogens that infect the honeybees. Several molecular techniques have been developed for the detection of pathogens like *Paenibacillus larvae*, *Melissococcus plutonius*, *Nosema ceranae* and *Nosema apis* [129, 146, 147], *Ascophera apis* and *Ascophera ceranae*, and *A*. *flavus* [129, 148]. Among them can highlight the simple PCR [149–151], NESTED-PCR [152], RT-PCR [153, 154], immunology-based tests (ELISA), and probe-based hybridization analysis [155]. The main advantages of these techniques would be less needed for sample treatment which often can be applied directly to the honeybee products, fast technique, specificity, and

The use of these techniques and the detection of this pathogen have allowed the control of mortality of honeybee populations around the world, restricting the dissemination of pathogens in bee products. For example, the diagnosis of American foulbrood and European foulbrood usually occurs through visual inspection of brood combs and detection of diseased larvae, subjective criteria that could be confused with other beehive conditions [155, 156]. The traditional methods of detection of these pathogens include the visualization by microscopy and detection in tissues [155]; culture on selective medium [151, 155, 156], including *P*. *larvae* agar [151]; bacteriophage sensitivity; immunotechniques; and microscopy of suspect bacterial strains have been considered adequate for routine identification purposes [151]; these methods are time-consuming and laborious but especially require that the infection is in progress so that the pathogen is detected and confirmed. The detection of pathogens before any clinical signs of disease to be visible in the colony would not only control these diseases but also the prevention of their consequences for the hive. That is, *M*. *plutonius* was detected in healthy colonies by RT-PCR in England and Wales, showing that the extent of

the prevalence of this pathogen in hives goes beyond the clinical signs [157].

antimicrobial molecules [140].

244 Honey Analysis

**7.2. Monitoring of the microbiological honey quality**

samples of Portugal [141, 142] and Argentina [119, 145].

**7.3. Detection of honeybee pathogens in honey**

sensitivity of detection.

Mayara Salgado Silva1 , Yavor Rabadzhiev2 , Monique Renon Eller1 , Ilia Iliev3 , Iskra Ivanova2 and Weyder Cristiano Santana4 \*

