Powdered Sugar Examination as a Tool for the Assessment of Paenibacillus larvae Infection Levels in Honey Bee Colonies.

American Foulbrood (AFB) is a contagious and severe brood disease of honey bees caused by the spore-forming bacterium Paenibacillus larvae. The identification of honey bee colonies infected by P. larvae is crucial for the effective control of AFB. We studied the possibility of identifying the infection levels by P. larvae in honey bee colonies through the examination of powdered sugar samples collected in the hives. The powdered sugar was dusted on the top bars of honeycombs and collected from a sheet paper placed at the bottom of the hive. Three groups of honey bee colonies were examined: Group A1- colonies with clinical symptoms of AFB (n = 11); Group A2 - asymptomatic colonies located in apiaries with colonies showing symptoms of AFB (n = 59); Group B - asymptomatic colonies located in apiaries without cases of the disease (n = 49). The results showed that there was a significant difference in spore counting between Groups and that the spore load in sugar samples was always consistent with the clinical conditions of the colonies and with their belonging to AFB-affected apiaries or not. Based on the obtained results the cultural examination of powdered sugar samples collected from hives could be an effective tool for the quantitative non-destructive assessment of P. larvae infections in honey bee colonies.

Combined Effects of Pesticides and Electromagnetic-Fields on Honeybees: Multi-Stress Exposure.

Honeybee and general pollinator decline is extensively reported in many countries, adding new concern to the general biodiversity loss. Many studies were addressed to assess the causes of pollinator decline, concluding that in most cases multi-stress effects were the most probable ones. In this research, the combined effects of two possible stress sources for bees, pesticides and electromagnetic fields (multi-stress conditions), were analyzed in the field. Three experimental sites were chosen: a control one far from direct anthropogenic stress sources, a pesticide-stress site and multi-stress one, adding to the same exposure to pesticides the presence of an electromagnetic field, coming from a high-voltage electric line. Experimental apiaries were monitored weekly for one year (from April 2017 to April 2018) by means of colony survival, queen activity, storage and brood amount, parasites and pathogens, and several biomarkers in young workers and pupae. Both exposure and effect biomarkers were analysed: among the first, acetylcholinesterase (AChE), catalase (CAT), glutathione S-transferase (GST) and alkaline phosphatase (ALP) and Reactive Oxygen Species (ROS); and among the last, DNA fragmentation (DNAFRAGM) and lipid peroxidation (LPO). Results showed that bee health conditions were the worst in the multi-stress site with only one colony alive out of the four ones present at the beginning. In this site, a complex picture of adverse effects was observed, such as disease appearance (American foulbrood), higher mortality in the underbaskets (common to pesticide-stress site), behavioral alterations (queen changes, excess of honey storage) and biochemical anomalies (higher ALP activity at the end of the season). The overall results clearly indicate that the multi-stress conditions were able to induce biochemical, physiological and behavioral alterations which severely threatened bee colony survival.

Phenotypic characterization and ERIC-PCR based genotyping of Paenibacillus larvae isolates recovered from American foulbrood outbreaks in honey bees from Italy.

BACKGROUND: Paenibacillus larvae is the etiological agent of American foulbrood (AFB), a widespread and severe bacterial brood disease of honey bees. The genomic characterization of P. larvae strains by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) is able to differentiate four genotypes (ERIC I, ERIC II, ERIC III, ERIC IV). The information on the presence of P. larvae ERIC genotypes worldwide is few. OBJECTIVES: We have characterized P. larvae strains isolated in Italy from AFB outbreaks to obtain information on ERIC genotypes and phenotypes of the strains circulating in the country. METHODS: A total of 117 P. larvae isolates from 115 AFB outbreaks occurring in 2008-2012 were subjected to phenotypic and genetic characterization. RESULTS: The genomic characterization allowed the identification of ERIC I and ERIC II genotypes. Examining the data of Northern and Central Italy separately it was noted that in Northern Italy most outbreaks were caused by the ERIC I genotype (78.6%), followed by the ERIC II genotype (18.6%) and by co-infections (ERIC I + ERIC II) (2.6%). In Central Italy, only outbreaks caused by the ERIC I genotype were observed. With regard to phenotypic characteristics all examined strains of ERIC II genotype fermented fructose while no strains of ERIC I genotype possessed this ability. CONCLUSION: Both P. larvae ERIC I and ERIC II genotypes were isolated from the AFB outbreaks, but ERIC II genotype was isolated only in Northern Italy. The fermentation of fructose seems to be a genotype-specific biochemical marker.

A new genotyping strategy for efficient scoring of closely positioned SNPs in the ovine prion protein gene.

Amino-acid polymorphisms of the ovine prion protein have been known to influence susceptibility to scrapie for many years. Recently, a role in both classical and atypical scrapie was assigned to new mutations, increasing the overall number of polymorphisms of interest for breeding plans. Besides, the high number and density of polymorphisms in the prion protein gene (PrP) and the presence of unusual mutations in some breeds hampers genotyping methods, making multiplexing difficult and sometimes compromising analytical results. We developed a multiplex genotyping method for the ovine PrP that overcomes the limitations posed by the high number and density of the polymorphisms to interrogate. Nine primers were designed to be compatible in a single primer-extension reaction developed for routine genotyping, with the capacity to identify the following polymorphisms: A136V, M137T, L141F, I142K, R154H, Q171R, Q171H, Q171K and N176K. Site-specific mutations were inserted in primer sequences in order to prevent extension of reciprocally complementary primers. Complete accuracy and repeatability of the assay was assessed with reference to 97 sequenced samples. The presented method constitutes an improved tool for ovine PrP genotyping and a general strategy for the use of primer extension in a genetic context of high density of polymorphisms.

Prion protein genotypes of Italian sheep breeds with lysine-171 and phenylalanine-141 detection.

Amino acid polymorphisms of the prion protein gene influence sheep susceptibility to classical and atypical scrapie. Substitutions at codons 136, 154 and 171 play an important role in classical scrapie. Codon 141 leucine to phenylalanine mutation (AFRQ) has been recognized as an increased risk factor for atypical scrapie. In addition a rare allele with lysine at codon 171 (ARK) has been detected in Mediterranean sheep breeds. The presence of ARK poses two problems: the determination of its frequency and its possible interference with genotyping output of routine methods lacking specific detection capacity for ARK. The aim of our work was the development of a routine genotyping method with the capacity to identify ARK and AFRQ in addition to the normally detected alleles and to determine the frequencies of all these alleles in 5 main Italian breeds: Sarda (n=2494), Bergamasca (n=2686), Appenninica (n=297), Comisana (n=361) and Massese (n=402). A multiplex primer extension assay targeting the six single nucleotide polymorphisms of interest was developed. Allele frequencies revealed a very low level of ARR in Bergamasca (6.91%) as opposed to the other breeds, very diverse levels of AFRQ ranging from absence in Comisana to 10.70% in Massese and a restricted presence of ARK. This allele has only been detected in Bargamasca with a significant 3.67% and marginally in Appenninica (0.34%). These results underline the need for adequate routine methods for genotyping of breeds with alleles that can interfere with typing of important codons such as the case of ARK for codon 171.