Maria Michalczyk

Detection of Nosema spp. in Worker Bees of Different Ages During the Flow Season

Abstract The aim of this study was to identify which Nosema species infect those Apis mellifera worker bees performing different functions in the colony. Samples were taken from different places inside and outside the hive, in the honey flow season. In February 2010, winter hive debris from 30 colonies was analyzed, and based on the microsporidian species identified by multiplex PCR. The following bee colonies (none of which displayed clinical symptoms of the disease) were selected for further analyses to determine the occurrence of microsporidian parasites: 1) colony A/C infected with Nosema apis and N. ceranae (mixed infection), 2) colony A infected with N. apis, 3) colony C - infected with N. ceranae, and 4) colony K - the control, which was free of infection. Between April and August, 20 nurse bees from frames of open brood, and 20 forager bees returning to the hive from pollen-collecting trips were randomly selected from each colony at 30-day intervals. The results of the study indicate that the microsporidian species is determined not only by the type of worker bee (sampling site), but also by the period (month) of the sample collection. Our findings also suggest that regardless of the type of initial infection, bees infected by different microsporidian species and bees free from infection can coexist in colonies. Streszczenie Celem badań było ustalenie, który gatunek Nosema występuje u robotnic Apis mellifera pobieranych z różnych miejsc ula w sezonie pasiecznym. W lutym 2010 roku zbadano osypy zimowe pszczół z 30 rodzin pszczelich. W zależności od stwierdzonego gatunku sporowca (metodą multiplex PCR) dalszą ocenę występowania gatunku sporowców prowadzono w następujących grupach (rodzinach pszczelich bez objawów klinicznych choroby): AC - zainfekowana Nosema apis i Nosema ceranae (infekcja mieszana), A - zainfekowana N. apis, C - zainfekowana N. ceranae oraz grupa kontrolna K, wolna od infekcji. Z każdej rodziny od kwietnia do sierpnia w odstępach 30 dniowych pobierano do badań losowo po 20 robotnic z dwóch miejsc: robotnice ulowe, bezpośrednio z plastrów z czerwiem otwartym oraz robotnice lotne - robotnice powracające do uli z pyłkiem. Wykazaliśmy, że nie tylko rodzaj robotnic (miejsce pobrania), ale i okres (miesiąc) pobrania prób do badań ma wpływ na rodzaj wykrywanego gatunku sporowca, a także, że niezależnie od rodzaju infekcji początkowej (w osypie zimowym) w rodzinach pszczelich mogą współistnieć osobniki zainfekowane różnymi gatunkami sporowca oraz wolne od infekcji.

Estimation of infection of internal parasites in horses from different type of farms.

Studies were carried out in year 2014 during the pasture period (from April to October) in Warmia and Mazury Region. Fecal samples were taken from cold- and warmblood horses from individual and agrotouristic farms with the different housing, feeding and pasture- care practices. Total of 512 horses were examined (320 mares, 170 geldings and 22 stallions). In the group of 185 horses from individual farms, 119 animals (64.3%) were infected with gastro-intestinal parasites. Among the 372 horses from agrotouristic farms 169 (51.7%) were infected with parasites. Most of the animals expelled the eggs of Cyathostominae. In some individuals occurred eggs of Strongylus spp., Parascaris equorum, Strongyloides westeri and tapeworm of Anoplocephala. The number of infected horses from agrotouristic farms was lower than from individual farms, probably due to more regular deworming (usually 2 times a year) and bigger care paid to cleaning pastures.

A study of single nucleotide polymorphism in the ystB gene of Yersinia enterocolitica strains isolated from various wild animal species

INTRODUCTION AND OBJECTIVE Y. enterocolitica is the causative agent of yersiniosis. The objective of the article was a study of single nucleotide polymorphism in the ystB gene of Y. enterocolitica strains isolated from various wild animal species. MATERIALS AND METHOD High-resolution melting (HRM) analysis was applied to identify single nucleotide polymorphism (SNP) of ystB gene fragments of 88 Y. enterocolitica biotype 1A strains isolated from wild boar, roe deer, red deer and wild ducks. RESULTS HRM analysis revealed 14 different melting profiles - 4 of them were defined as regular genotypes (G1, G2, G3, G4), whereas 10 as variations. 24 of the examined Y. enterocolitica strains were classified as G1, 18 strains as a G2, 21 strains as a G3, and 15 strains as a G4. Nucleotide sequences classified as G1 revealed 100% similarity with the Y. enterocolitica D88145.1 sequence (NCBI). Analysis of G2 revealed one point mutation - transition T111A. One mutation was also found in G3, but SNP was placed in a different gene region - transition G193A. Two SNPs - transitions G92C and T111A - were identified in G4. Direct sequencing of 10 variations revealed 5 new variants of the ystB nucleotide sequence: V1 - transition G129A (3 strains); V2 - transitions T111A and G193A (2 strains); V3 - transitions C118T and G193A (1 strain); V4 - transitions C141A and G193A (2 strains); and V5 characterized by 19 SNPs: G83A, T93A, A109G, G114T, C116T, A123G, T134C, T142G, T144C, A150C, G162A, T165G, T170G, T174A, T177G, G178A, A179G, A184G and G193A (2 strains). The predominant genotype in isolates from wild ducks was G1; in red deer G2; in wild boar G3; in roe deer G1 and G4. CONCLUSIONS The proposed HRM method could be used to analyze Y. enterocolitica biotype 1A strains isolated from different sources, including humans.

Characterisation of ail-positive Yersinia enterocolitica of different biotypes using HRMA

Yersiniosis is one of the four most frequent foodborne zoonotic diseases in Europe, and Yersinia enterocolitica is the primary agent in human infections. The ail gene is an important chromosomal virulence marker of Y. enterocolitica which encodes Ail, a 17-kDa outer membrane protein that promotes attachment and invasion. In the present study, ail-positive Y. enterocolitica strains of different biotypes were examined using high resolution melting analysis (HRMA) and DNA sequencing. Genotype data relating to Y. enterocolitica strains isolated from different sources and belonging to different biotypes were compared. Applied method allowed efficient distinguishing of three genotypes and phylogenetic groups: 1A - included non-pathogenic Y. enterocolitica strains; 1B - consisted of highly pathogenic Y. enterocolitica strains and 2/4 - involved weakly pathogenic Y. enterocolitica strains. Amplicon genotyping based on HRMA supports rapid identification of ail SNPs correlated with biotype of examined Y. enterocolitica strains.

Biochemical status of feral honey bees (Apis mellifera) infested with various pathogens

Feral bees colonizing large forest habitats without human intervention are also affected by diseases. Infected bees that survive several seasons constitute an interesting object of scientific inquiry. Little is known about the health and status of feral honey bee populations. The pathogens infesting/infecting bee colonies and the biochemical status of unhealthy bees need to be studied to prevent the global decline in bee populations. Honey bees inhabiting hollow trees in forest reserves of north-eastern Poland, infected with Nosema ceranae or infested with Varroa destructor and/or infected with the deformed wing virus (DWV), were analyzed. Differences in the analyzed biochemical parameters were noted in groups infected/infested with various pathogens relative to the control group. The most significant variations were observed in antioxidant parameters: lower total antioxidant status (TAS) (excluding group N. ceranae), lower activity of superoxide dismutase (SOD) and peroxidase (POX) (excluding group N. ceranae + DWV), higher activity of glutathione transferase (GST), and higher glutathione (GSH) levels in groups infected with N. ceranae. Glycogen concentration was higher in DWV-infected bees. In comparison with bees infested with V. destructor only, co-infested groups were characterized by fluctuations in carbohydrate concentration, subject to the co-infesting pathogen. The presence of a single pathogen has a less detrimental effect on most metabolic parameters in feral honey bees than concomitant infections with several pathogens. The results of this study suggest that co-infestations with V. destructor and DWV in feral honey bees have more serious consequences than co-infections with Nosema sp. and DWV.

Evaluation of the Effectiveness of Selected Treatments of Nosema Spp. Infection by the Hemocytometric Method and Duplex Pcr

Abstract Recent years have witnessed an increase in the mortality of honey bees in many regions of the world. The observed decrease in the bee population results from a combination of factors, and microsporidian parasites Nosema apis and N. ceranae are among the main contributors. Those parasites cause a microsporidian infection that shortens the lifespan of bees and reduces the productivity of bee colonies. The aim of this study was to evaluate the effectiveness of Nozevit, Api Herb and ApiX (acetylsalicylic acid + Artemisia absinthium L. extract) in the control of infections caused by Nosema spp. in a field experiment. Two groups of worker bees were evaluated - hive bees and forager bees returning to the hive. The effect of the analyzed therapies on the number of spores and the microsporidia species were analyzed by the hemocytometric method and duplex PCR. A statistical analysis revealed that the applied treatments had reduced the number of spores by 31.15% on average. In hive bees, Nosema spp. infection was most effectively reduced by Nozevit (67.85%) and ApiX (63.36%). Coinfections (N. ceranae and N. apis) were affirmed in all bee samples before treatments. However, after the treatments, single infection of N. apis and N. ceranae were detected. The tested treatments were more effective in the control of N. apis than N. ceranae.

Estimation of the influence of selected products on co-infection with N. apis/N. ceranae in Apis mellifera using real-time PCR

Abstract To protect the world’s honey bee population many scientific centres are searching for products and methods that control nosemosis. Real-time PCR was used to assess infection level in worker bees infected with Nosema spp. in bee colonies co-infected with Nosema apis and Nosema ceranae after the administration of three products (Nozevit, ApiHerb and ApiX) and sugar syrup. The study was conducted in the field condition therefore there was no possibility to affect the number of spores in the selected material. The study demonstrated considerable differences in the number of spores of individual Nosema spp. in the analysed samples of bees. HSD Tukey’s test showed that the statistically significant effect on limiting the N. apis invasion had ApiX (p – 0.049). Nozevit, Apiherb and syrup showed no statistically significant effect on reducing the amount of N. apis spores. The same test showed that the statistically significant effect on limiting the N. ceranae invasion had: Nozevit (p – 0.014), Apiherb (p – 0.032), ApiX (p – 0.034) and syrup (p – 0.033). There was no statistically significant decrease in the N. ceranae spores in the control group.