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.
Within the genus
Trichinella, Trichinella pseudospiralis
is the only recognized non-encapsulated species known to infect mammals and birds. In October 2020, larvae recovered from muscle tissues of a wolf (
Canis lupus italicus
) originating from Molise Region, Central Italy, were molecularly confirmed as those of
Trichinella britovi
and
T. pseudospiralis
. This is the first detection of
T. pseudospiralis
from a wolf. In Italy, this zoonotic nematode was detected in a red fox (
Vulpes vulpes
), three birds (
Strix aluco
,
Athene noctua
,
Milvus milvus
) and five wild boars (
Sus scrofa
), and was also identified as the etiological agent of a human outbreak of trichinellosis in 2015. Since
T. pseudospiralis
is rarely reported from carnivore mammals in comparison to the encapsulated species frequently detected in these hosts, this finding opens the question of the role of carnivores as reservoirs for this parasite.
In the summer of 2009, high levels of mortality among white clawed crayfish Austropotamobius pallipes were observed in 3 watercourses of central Italy. PCR and culture methods were used to detect the causative agent of the disease. Two strains of Aphanomyces spp. were isolated and identified by PCR and DNA sequencing as Aphanomyces astaci and A. repetans. This is the first crayfish plague outbreak in Italy to be confirmed by the isolation in culture of a pathogen from Austropotamobius pallipes.
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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