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.

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.

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.

The detection/quantification in honey of spores of the bacterial pathogen Paenibacillus larvae, etiological agent of the American Foulbrood (AFB) infectious disease of honey bees, represents a useful diagnostic tool to identify apiaries at risk of outbreaks and apply prevention measures. Plate count is the method in use for analyzing presence and number of spores but its validity needs to be re-assessed since P. larvae field strains showed a low rate of germination in culture media. To this aim, culture-dependent P. larvae detection/quantification was compared in this study with quantitative PCR (qPCR) based analysis for 139 honey samples, each from a different apiary, collected in 2017 and 2018 in the Abruzzo region. According to qPCR based detection, 59.6% samples contained P. larvae spores, while, according to plate count, only 32.3% samples were contaminated. Moreover, the levels of contamination determined with the two methods differed. Many samples (29.5%) were negative with the cultivation assay but positive with the qPCR test, suggesting that not all the field strains were able to develop in plate. This hypothesis was confirmed by re-testing those samples with PLA medium added with germination stimulants and obtaining P. larvae growth. Results strongly suggested the necessity to apply improved culture methods or molecular detection/quantification for a more reliable AFB risk estimation in order to efficiently fight this honey bee plague.

Phoridae, which belong to the Diptera order, were identified as the cause of serious infestations of Apis mellifera colonies in the American continent such as Phoridae from the genus “Melaloncha” in Central America and Apocephalus borealis in the USA. Some authors identified a novel phorid, Megaselia scalaris, as a possible parasitoid of honey bee in Brazil. Recently, in Europe, Diptera from the genus Megaselia was found able to parasitize adults of A. mellifera. So far, in the old continent, such infestation was reported only in bees dead or affected by other diseases, but never in healthy bees. From July through August 2014, the prevalence of myiases affecting A. mellifera was verified in Abruzzo and Molise regions. Most of the selected apiaries showed a great infestation of bees due to M. scalaris, even if they were apparently healthy, during trapping back from their foraging trip. Here, we report the results of our work to stress the impact that this parasitoid might have on health condition of beehive.

American foulbrood (AFB) of Apis mellifera, caused by the bacterial spore former Paenibacillus larvae, is a notifiable disease in Italy and its spread is prevented by burning hives with clinical symptoms. Official epidemiological data indicate a very low number of annual outbreaks in the country, therefore this investigation was carried out to evaluate whether notification to the veterinary authority is omitted, so that AFB transmission from infected apiaries is not prevented. Presence and number of P. larvae spores were analyzed in 122 honey samples, each from a different apiary, collected in the Abruzzo region in years 2014 and 2015. P. larvae spores were found in 40% of the samples and the spore numbers allowed to infer a prevalence of AFB of at least 22.7% among beekeepers, indicating the necessity to improve the surveillance system. Spore numbers were significantly different for honeys produced in different environmental contexts and with varying management practices, suggesting to elucidate the causal relationship between those factors and the presence of P. larvae spores.