PCR-Based Screening of Pathogens in Bombus terrestris Populations of Turkey.

PURPOSE: Bumblebees are an important group of insects in the pollination of various vegetables, fruits, oilseeds, legumes, and the fodder crops. Compared to honeybees, they have a wider choice of hosts and a longer flight period. These bees are used especially for the pollination of plants in greenhouses and are commercially produced for this purpose. Recently, serious decreases have been occurring in bumblebee populations due to various reasons such as pathogens, and some of species are even threatened with extinction. Due to the worldwide decline in pollinator insects, determining the distribution and prevalence of bumblebee pathogens is of great importance. Therefore, this study was conducted to determine the incidence and prevalence of pathogens in Turkish bumblebee populations and how much of each pathogen was in bumblebee samples. METHODS: A total of 172 Bombus terrestris (Linnaeus,1758) samples (21 samples from commercial enterprises, 79 samples from greenhouses and 72 samples from nature) were randomly collected from 3 provinces (Antalya, Mersin and Izmir) where greenhouse cultivation is intensively carried out in Turkey. Eighty-nine of these samples were collected in the spring and eighty-three in the autumn. The presence of four pathogens (Nosema bombi, Crithidia bombi, Apicystis bombi, and Locustacarus buchneri) was investigated by PCR using universal primers. RESULTS: The overall prevalence of Nosema bombi, Crithidia bombi, Apicystis bombi, and Locustacarus buchneri was determined as 7.55%, 9.3%, 11.62%, and 4.65%, respectively. Co-infections (5.81%) were only detected in wild-caught (nature) samples. C. bombi and A. bombi infections were detected at higher rates in the spring samples than in the autumn samples (p < 0.05). There was no significant difference between the spring and autumn samples with respect to the presence of N. bombi and L. buchneri (p > 0.05). CONCLUSION: The results obtained could be important in determining the prevalence and spread rates of the bumblebee diseases in Turkey and to determine appropriate protection measures. The information gathered should increase our knowledge about the presence of these pathogens in Turkey and could contribute to improve apiarist's practice. More studies are needed to determine the transmission pathways of these pathogens between the populations. Also, complex pathogen interactions in bumblebee populations should be considered in the future to improve bumblebee health.

Molecular investigation of Coxiella burnetii in the Middle and East Black Sea region in aborted bovine fetuses and investigation of the oxidant/antioxidant system.

Coxiella burnetii (C. burnetii) is a causative microorganism that causes the zoonotic Q fever disease, which is generally asymptomatic in animals, but causes reproductive issues such as abortion, stillbirth, and infertility. C. burnetii infection poses a threat to farm economies as it affects productivity in farm animals. The purpose of this research was to look into the incidence of Q fever in eight provinces in the Middle and East Black Sea region and to measure reactive oxygen and reactive nitrogen species as well as antioxidant levels in bovine aborted fetal livers infected with C. burnetii. The study material consisted 670 bovine aborted fetal liver samples delivered to Samsun Veterinary Control Institute from eight provinces between 2018 and 2021. C. burnetii was analyzed by PCR in these samples and 47 (7,01%) were positive while 623 negative. Nitric oxide (NO), malondialdehyde (MDA) and reduced glutathione (GSH) activities were analyzed by spectrophotometric method in both 47 positive samples and 40 negative samples as control group. In the C. burnetii positive and control groups, MDA levels were determined to be 2.46 ± 0.18 and 0.87 ± 0.07 nmol/ml, NO levels were determined to be 1.77 ± 0.12 and 1.09 ± 0.07 nmol/ml, and reduced GSH activity was determined to be 5.14 ± 0.33 and 6.62 ± 0.46 µg/dl, respectively. In C. burnetii positive fetal liver tissue, MDA and NO levels were higher than the control group, while reduced GSH levels were lower than the control group. As a result, C. burnetii caused changes in free radical level and antioxidant activity in bovine aborted fetus liver.

PCR-based detection of the honeybee tracheal mite (Acarapis woodi) in Türkiye.

Acarapis woodi (Rennie 1921) (Acari: Tarsonemidae) is one of the mites that settles in the respiratory system of honeybees (Apis mellifera L. (Hymenoptera, Apidae)) and distributed throughout the world. It causes significant economic losses on honey production. In Türkiye, studies on the existence of A. woodi are very limited and so far, no studies on the molecular diagnosis and phylogenetic of it have been reported in Türkiye. This study was conducted to investigate the prevalence of A. woodi in Türkiye, especially in areas where beekeeping is intense. Diagnosis of A. woodi was performed using both microscopic and molecular methods using specific PCR primers. Adult honeybee samples were collected from 1.193 hives in 40 provinces of Türkiye between 2018 and 2019. Based on identification studies, the presence of A. woodi was detected in a total of 3 hives (0.5%) in 2018 and 4 hives (0.7%) in 2019. This is the first report for determination of A. woodi in Türkiye.

Use of Dicranum polysetum extract against Paenibacillus larvae causing American Foulbrood under in vivo and in vitro conditions.

Recent research shows that Dicranum species can be used to ameliorate the negative effects of honeybee bacterial diseases and that novel compounds isolated from these species may have the potential to treat bacterial diseases. This study aimed to investigate the efficacy of Dicranum polysetum Sw. against American Foulbrood using toxicity and larval model. The effectiveness of D. polysetum Sw. ethanol extract in combating AFB was investigated in vitro and in vivo. This study is important in finding an alternative treatment or prophylactic method to prevent American Foulbrood disease in honey bee colonies. Spore and vegetative forms of Paenibacillus larvae PB31B with ethanol extract of D. polysetum were tested on 2040 honey bee larvae under controlled conditions. Total phenolic and flavonoid contents of D. polysetum ethanol extracts were determined as 80.72 mg/GAE(Gallic acid equivalent) and 303.20 µg/mL, respectively. DPPH(2,2-diphenyl-1-picrylhydrazyl) radical scavenging percent inhibition value was calculated as 4.32%. In Spodoptera frugiperda (Sf9) and Lymantria dispar (LD652) cell lines, the cytotoxic activities of D. polysetum extract were below 20% at 50 µg/mL. The extract was shown to considerably decrease infection in the larvae, and the infection was clinically halted when the extract was administered during the first 24 h after spore contamination. The fact that the extract contains potent antimicrobial/antioxidant activity does not reduce larval viability and live weight, and does not interact with royal jelly is a promising development, particularly regarding its use to treat early-stage AFB infection.

Phytochemicals, antimicrobial, and sporicidal activities of moss, Dicranum polysetum Sw., against certain honey bee bacterial pathogens.

Beekeeping is an important agricultural and commercial activity globally practiced. Honey bee is attacked by certain infectious pathogens. Most important brood diseases are bacterial including American Foulbrood (AFB), caused by Paenibacillus larvae (P. larvae), and European Foulbrood (EFB) by Melissococcus plutonius (M. plutonius) in addition of secondary invaders, e.g. Paenibacillus alvei (P. alvei) and Paenibacillus dendritiformis (P. dendritiformis). These bacteria cause the death of larvae in honey bee colonies. In this work, antibacterial activities of extracts, fractions, and isolated certain compounds (nominated 1-3) all originated from moss, Dicranum polysetum Sw. ( D. polysetum), were tested against some honey bee bacterial pathogens. Minimum inhibitory concentration, minimum bactericidal concentration, and sporicidal values ​​of methanol extract, ethyl acetate, and n-hexane fractions ranged between 10.4 and 18.98, 83.4-303.75 & 5.86-18.98 µg/mL against P. larvae, respectively. Antimicrobial activities of the ethyl acetate sub-fractions (fraction) and the isolated compounds (1-3) were tested against AFB- and EFB-causing bacteria. Bio-guided chromatographic separation of ethyl acetate fraction, a crude methanolic extract obtained from aerial parts of D. polysetum resulted in three natural compounds: a novel one, i.e. glycer-2-yl hexadeca-4-yne-7Z,10Z,13Z-trienoate (1, dicrapolysetoate; given as trivial name), in addition to two known triterpenoids poriferasterol (2), and γ-taraxasterol (3). Minimum inhibitory concentration ranges were 1.4-60.75, 8.12-65.0, 2.09-33.44 & 1.8-28.75 µg/mL for sub-fractions, compounds 1, 2, and 3, respectively.

Honeybee-associated lactic acid bacteria and their probiotic potential for human use.

This study aims to identify lactic acid bacteria (LAB) isolated from honeybees (Apis mellifera workers and larvae) in detail and to determine their functional probiotic properties. A total of 11 strains were classified based on morphological and biochemical characteristics. Preliminary probiotic properties of strains, that were molecularly identified using 16 S rRNA, such as antimicrobial activity, tolerance to digestive conditions, aggregation ability, were investigated. The antimicrobial properties of strains were tested against a wide range of human pathogens. All strains that showed γ-hemolysis and did not contain bacteriophages were considered safe. The strains' survivability checked for 0.3% bile and 3.0-7.8 pH contents was promising. The highest autoaggregation ranged from 14.7 to 30.76% after 4 h. Tested LAB strains markedly exhibited coaggregation with Listeria monocytogenes and Escherichia coli. According to the results, tested bacteria showed significant antagonistic effects against pathogens, and positive probiotic characteristics compatible with in vitro gastrointestinal tract conditions. The results suggest that Apis mellifera LAB symbionts may have a probiotic potential, and be effective and safe candidates for human use. This study provides an addition to the development of the current knowledge by defining in detail honeybee-associated bacteria and determining their probiotic potential.

Biological Activity and Phytochemical Analysis of Dicranum scoparium against the Bacterial Disease for Honey Bee.

Bacterial diseases, such as American Foulbrood (AFB) and European Foulbrood (EFB), are known to have catastrophic effects on honey bees (if left to spread, can wipe out entire colonies), leading to severe financial losses in the beekeeping industry. The aim of this study was to evaluate the pharmacological properties of methanol extract and its fractions (ethyl acetate, hexane, water) derived from Dicranum scoparium Hedw., which could be utilized as a potential drug to prevent the bacterial diseases (AFB and EFB) affecting the honey bees. For this purpose, crude methanol extract and ethyl acetate/hexane/water fractions were prepared from the aerial part of D. scoparium, collected from Trabzon province. Bio-guided fractionation of the extract and its fractions led to the first-time isolation of five compounds. The structure of all compounds was elucidated by nuclear magnetic resonance (NMR) spectroscopy, ultraviolet (UV) spectral analysis, Fourier-transform infrared spectroscopy (FT-IR), liquid chromatography quadrupole time-of-flight mass spectroscopy (LC-QToF-MS), and by comparison of their NMR data with that of literature. The analysis of these compounds revealed significant antibacterial and sporicidal activities against bacteria causing larval diseases in honey bees. The antibacterial activity of these compounds ranged from 0.6 to 60 µg/mL against AFB and EFB causing bacteria. Therefore, the natural raw extract and fractions of D. scoparium could be used as potential therapeutic agents against bacterial agents affecting honey bees.

Isolation, characterization, and comparative genomic analysis of vB_PlaM_Pd22F, a new bacteriophage of the family Myoviridae.

The use of phage and phage-based products for the prevention and treatment of bee disease is one of the promising natural alternatives to chemical or antibiotic treatments in beekeeping. A novel lysogenic bacteriophage, phage Pd22F (vB_PlaM_Pd22F), was isolated from Paenibacillus dendritiformis by the prophage induction method. This phage, which is capable of infecting Paenibacillus larvae and P. dendritiformis strains, was characterized by microbiological and comparative genomic analysis. Transmission electron microscopy images showed that phage Pd22F had the morphology of a myovirus. Whole-genome sequencing results showed that vB_Pla M_Pd22F has an 86,388-bp linear dsDNA genome with a GC content of 50.68%. This genome has 124 coding sequences (CDSs), 53% of which encode functionally unknown proteins and 57 of which encode proteins that show similarity to known proteins. In addition, one tRNA gene was found. The phage Pd22F genome does not contain any antimicrobial resistance genes. The similarity between the genome sequence of phage Pd22F and the whole genome sequences of other Paenibacillus phages available in the NCBI Virus Database was found to be below 50% (42%), indicating that phage Pd22F differs greatly from previously characterized phages at the DNA level. The results of comparative genomics and phylogenetic analysis revealed that Pd22F is a new phage belonging to the family Myoviridae, order Caudovirales. This is the first report of genomic and morphological characterization of a Paenibacillus dendritiformis prophage.

Isolation, characterization, and comparative genomic analysis of vB_PlaP_SV21, new bacteriophage of Paenibacillus larvae.

Paenibacillus larvae cause an American foulbrood disease (AFB) that is responsible for the extinction of honeybee colonies and is a honeybee bacterial disease that has to be obligatory notified worldwide. Recently, bacteriophage studies targeting Paenibacillus larvae have emerged as a promising alternative treatment method. The inability of bacteria to create resistance against bacteriophages makes this method advantageous. As a consequence, this study was conducted to describe the genome and biological characteristics of a novel phage capable of lysing Paenibacillus larvae samples isolated from honeybee larva samples in Turkey. The Paenibacillus phage SV21 (vB_PlaP_SV21) was isolated by inducing Paenibacillus larvae strain SV21 with Mitomycin-C. Whole-genome sequencing, comparative genomics, and phylogenetic analysis of vB_PlaP_SV21 were performed. Transmission electron microscopy images showed that vB_PlaP_SV21 phage was a Podovirus morphology. The vB_PlaP_SV21 phage specific for Paenibacillus larvae was determined to belong to the Podoviridae family. Host range and specificity, burst size, lytic activity, and morphological characteristics of the phage were determined. Bioinformatic analysis of the Paenibacillus phage SV21 showed 77 coding sequences in its linear 44,949 bp dsDNA genome with a GC content of 39.33%. In this study, we analysed the genomes of all of the currently sequenced P. larvae phage genomes and classified them into five clusters and a singleton. According to molecular, morphological, and bioinformatics results, it was observed that API480 (podovirus), which was reported as a singleton in previous studies and public databases, and Paenibacillus phage SV21 phage could form a new cluster together.

Biocidal properties of maltose reduced silver nanoparticles against American foulbrood diseases pathogens.

Bee disease caused by spore-forming Paenibacillus larvae and Paenibacillus alvei is a serious problem for honey production. Thus, there is an ongoing effort to find an effective agent that shows broad biocidal activity with minimal environmental hazard. In this study, the biocidal effect of maltose reduced silver nanoparticles (AgNPs) is evaluated against American foulbrood and European foulbrood pathogens. The results demonstrate that the maltose reduced AgNPs are excellent short and long-term biocides against P. larvae isolates. The long-term effect suggests that the Ag+ ions are released from the AgNPs with increasing time in a controlled manner.