A study in the Abruzzo region on the presence of <i>Paenibacillus larvae</i> spores in honeys indicated underestimation of American foulbrood prevalence in Italy

Ricchiuti, Luciano; Rossi, Franca; Matto, Ilaria Del; Iannitto, Giorgio; Riccio, Angela Lucia del; Petrone, Domenico; Ruberto, Giovanni; Cersini, Antonella; Domenico, Marco Di; Cammà, Cesare

doi:10.1080/00218839.2018.1541651

“…In comparison to a previous study carried out three years earlier in the same geographical area (Ricchiuti et al, 2019) by using non-supplemented PLA medium, the percentage of positive samples was similar.…”

Section: Resultssupporting

confidence: 69%

“…Detection/enumeration by qPCR indicated an even higher prevalence, thus showing that not much was done to contrast AFB spread in a region where apiculture is largely practiced (Ricchiuti et al, 2019). This situation is particularly serious, considering that even for low levels of honey contamination AFB clinical signs can occur (Bassi et al, 2018).…”

Section: Resultsmentioning

confidence: 99%

Quantitative PCR (qPCR) vs culture-dependent detection to assess honey contamination by Paenibacillus larvae

Crudele

¹

,

Ricchiuti

²

,

Ruberto

³

et al. 2019

Preprint

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The detection/quantification in honey of spores of the bacterial pathogen Paenibacillus larvae, the etiological agent of the American Foulbrood (AFB) infectious disease of honey bee, represents a useful diagnostic tool to identify apiaries at risk of infection and carry out focused prevention measures.Plate count is currently the recommended method used to analyze presence and number of spores for this pathogen. However, its validity needs to be re-assessed since P. larvae field strains have a low rate of germination in culture media.Therefore, in this study, culture-dependent P. larvae detection/quantification was compared with quantitative PCR (qPCR) based analysis for 139 honey samples collected in 2017 and 2018 from as many apiaries in the Abruzzo region. According to qPCR based detection, 58.27% samples were positive for P. larvae, while only 33.8 % samples were positive by plate count.Moreover, the levels of contamination with the two methods differed for most samples. Potential false positives were obtained by plate count for 12.9% samples that were negative with the qPCR test. On the other hand, 26.6% samples were culture negative but qPCR positive, suggesting that not all the field strains were able to develop in plate. This was confirmed by obtaining P. larvae growth from those samples after supplementing the medium with germination stimulants.Results strongly suggested the necessity to improve culture methods or replace them with molecular detection/quantification for a more reliable AFB risk estimation.

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“…In the European countries antibiotic use for AFB treatment is banned and the spread of P. larvae spores is prevented exclusively by burning infected hives and equipment after manifestation of the disease symptoms, a tardive, thus inefficient, measure (Locke et al, 2019). Moreover, a study in the Abruzzo region suggested that the latter practice, though being mandatory by law, is largely disregarded (Ricchiuti et al, 2019). Examination of hive matrices such as honey (Alippi et al, 2004), hive debris (Bassi et al, 2018) and adult bees (Forsgren and Laugen, 2014) for the presence of P. larvae spores can allow early diagnosis before clinical manifestation and prevents P. larvae massive multiplication and transmission (OIE, 2019).…”

Section: Introductionmentioning

confidence: 99%

Quantitative PCR (qPCR) vs culture-dependent detection to assess honey contamination by Paenibacillus larvae

Crudele

¹

,

Ricchiuti

²

,

Ruberto

³

et al. 2019

Journal of Apicultural Research

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The detection/quantification in honey of spores of the bacterial pathogen Paenibacillus larvae, the etiological agent of the American Foulbrood (AFB) infectious disease of honey bee, represents a useful diagnostic tool to identify apiaries at risk of infection and carry out focused prevention measures.Plate count is currently the recommended method used to analyze presence and number of spores for this pathogen. However, its validity needs to be re-assessed since P. larvae field strains have a low rate of germination in culture media.Therefore, in this study, culture-dependent P. larvae detection/quantification was compared with quantitative PCR (qPCR) based analysis for 139 honey samples collected in 2017 and 2018 from as many apiaries in the Abruzzo region. According to qPCR based detection, 58.27% samples were positive for P. larvae, while only 33.8 % samples were positive by plate count.Moreover, the levels of contamination with the two methods differed for most samples. Potential false positives were obtained by plate count for 12.9% samples that were negative with the qPCR test. On the other hand, 26.6% samples were culture negative but qPCR positive, suggesting that not all the field strains were able to develop in plate. This was confirmed by obtaining P. larvae growth from those samples after supplementing the medium with germination stimulants.Results strongly suggested the necessity to improve culture methods or replace them with molecular detection/quantification for a more reliable AFB risk estimation.

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“…The method of DNA extraction and the qPCR assay with primers PLAup/PLAdw were applied to analyze hive debris, honey from a honey comb, adult bees and brood cells with larval scales from two AFB outbreaks occurring in two experimental apiaries. Moreover, this method was used to analyze DNA samples extracted from 50 field strains identified in a previous study [19]. With no exception, positive results were obtained from all these sources.…”

Section: Resultsmentioning

confidence: 99%

“…Reference bacterial strains used in this study were P. larvae ATCC 9545, P. naphthalenovorans DSM 14203, P. glucanolyticus DSM 5162 and P. chitinolyticus DSM 11030. In addition, 50 P. larvae isolates previously identified with the end point PCR test with primers AFB-F/AFB-R [19], were used to experimentally confirm the inclusivity of the new test. All the strains were grown on Paenibacillus larvae agar (PLA), in which all the Paenibacillus species tested grew well, prepared as described by Schuch et al [20] with components from Sigma Aldrich (Milan, Italy), or on Sheep Blood Agar (Biolife Italiana, Milan, Italy) incubated at 37 °C for 2–5 days in the presence of 9% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Quantitative PCR (qPCR) Paenibacillus larvae Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris

Rossi

¹

,

Amadoro

²

,

Ruberto

³

et al. 2018

Insects

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The application of quantitative PCR (qPCR) as a routine method to detect and enumerate Paenibacillus larvae in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of Apis mellifera. However, none of the qPCR tests described so far has been officially proposed as a standard procedure for P. larvae detection and enumeration for surveillance purposes. Therefore, in this study, inclusivity, exclusivity and sensitivity of detection of P. larvae spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim, recently acquired P. larvae sequence data were considered to assess inclusivity in silico and more appropriate non-target species were used to verify exclusivity experimentally. This led to the modification of a previously described method by shortening the forward primer, designing a new reverse primer and using more stringent amplification conditions. The new test allowed the detection of P. larvae spores in honey and hive debris down to 1 CFU/g. The qPCR test optimized in this study proved suitable for quantification and also for identification of field P. larvae strains and real contaminated samples. Therefore, it is proposed for reliable detection and quantification of P. larvae in honey and hive debris, thus circumventing the disadvantages of late AFB diagnosis based on clinical symptoms and possible underestimation of spore numbers that is the main drawback of culture-dependent procedures.

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A study in the Abruzzo region on the presence of Paenibacillus larvae spores in honeys indicated underestimation of American foulbrood prevalence in Italy

Cited by 7 publications

References 13 publications

Quantitative PCR (qPCR) vs culture-dependent detection to assess honey contamination by Paenibacillus larvae

Quantitative PCR (qPCR) vs culture-dependent detection to assess honey contamination by Paenibacillus larvae

Quantitative PCR (qPCR) vs culture-dependent detection to assess honey contamination by Paenibacillus larvae

Evaluation of Quantitative PCR (qPCR) Paenibacillus larvae Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris

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