Selection and Multiplexing of Reverse Transcription-Quantitative PCR Tests Targeting Relevant Honeybee Viral Pathogens.

Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription-quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction.

Recent Trends of Antibiotic Resistance in Staphylococcus aureus Causing Clinical Mastitis in Dairy Herds in Abruzzo and Molise Regions, Italy.

This study aimed to investigate the recent trends of antibiotic resistance (AR) prevalence in Staphylococcus aureus isolated from the milk of animals with clinical mastitis in areas of the Abruzzo and Molise regions in Central Italy. Fifty-four S. aureus isolates were obtained from routine testing for clinical mastitis agents carried out in the author institution in the years 2021 and 2022 and were analyzed for phenotypic resistance to eight antibiotics recommended for testing by European norms and belonging to the antibiotic classes used for mastitis treatment in milk-producing animals. Moreover, the presence of 14 transferable genetic determinants encoding resistance to the same antibiotics was analyzed using qPCR tests developed in this study. Phenotypic resistance to non-ß-lactams was infrequent, with only one 2022 isolate resistant to clindamycin. However, resistance to the ß-lactam cefoxitin at concentrations just above the threshold of 4 µg/mL was observed in 59.2% of isolates in both years, making these isolates classifiable as methicillin-resistant. The AR genotypes detected were the blaZ gene (50% of 2021 isolates and 44.4% of 2022 isolates), aphA3-blaZ- ermC/T (one 2021 isolate), aphA3-ant6-blaZ-ermC/T (one 2021 isolate), blaZ-ermB (one 2022 isolate) and mecA-mph (one 2022 isolate). An inquiry into the veterinarians who provided the samples, regarding the antimicrobials prescribed for mastitis treatment and criteria of usage, indicated a possible causal relation with the AR test results. The occurrence of AR genotypes did not increase in time, most probably reflecting how mastitis was treated and prevented in farms. However, the frequently observed cefoxitin resistance needs to be explained genotypically, further monitored and limited by modifying antibiotic usage practices. The identification of a mecA-positive isolate in 2022 suggests further investigation if this genotype is emerging locally.

First report of Trichinella pseudospiralis in a wolf (Canis lupus italicus).

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.

Contamination of honey by oxytetracycline from pig manure.

Although the use of antimicrobial is not allowed in bee industry according to current EU legislation, antimicrobial residues are often detected in honey doomed to human consumption. This study aims to investigate if bees living in hives located nearby tanks filled with pig manure containing residues of oxytetracycline, would naturally harvest water from it, thus contaminating their honey. Data from this experiment were compared with those originating from direct contamination with oxytetracycline through the beehive feeders. Bees did not harvest water from manure, even during the warmest days of summer. Instead, antimicrobial residues were evidenced and quantified in honey from hives directly contaminated with oxytetracycline. Interestingly, antimicrobial residues were also observed in honey from untreated hives thus suggesting that illegal treatments can cause contamination, albeit at low levels, of honey produced in legally-untreated neighboring hives.

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

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.

Confirmation of crayfish plague in Italy: detection of Aphanomyces astaci in white clawed crayfish.

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.