Isolation, characterization, and genetic diversity of Paenibacillus larvae from AFB suspected specimens in the Central and Eastern Black Sea Regions

American foulbrood is an important bacterial disease affecting the larvae of honey bees (Apis mellifera L.) caused by Paenibacillus larvae. Due to easy transmission of disease and the ability of bacteria to create spores, it is a bacterium resistant to both physical and chemical conditions. The study aims to isolate, perform microbiological analyses, and determine biochemical properties and genotypes P. larvae strains from AFB samples collected from Turkey's Central and Eastern Black Sea regions. An isolation study was conducted on adult bees, larvae, honey, and primary honeycomb samples from suspected colonies in the regions under study. After the purification of bacterial isolates from samples, P. larvae strains were identified using biochemical and molecular methods. The genetic diversity and ERIC types of P. larvae isolates were determined by rep-PCR DNA genotyping using BOX A1R and MBO REP1 primers and multiplex-PCR. A phylogenetic tree of P. larvae strains was constructed in the study. All P. larvae isolates were determined as ERIC I type. According to the rep-PCR results of P. larvae strains, 15 of the 28 isolates were Ab genotype (54%), 7 (25%) Aβ genotype, 4 (14%) AB genotype, 1 (3.5%) αB genotype, and 1 (3.5%) ab genotype. From an epidemiological viewpoint, it was determined that Ab and Aβ genotypes were widely distributed, while other genotypes (AB, αB, and ab) showed less spread. The results of the study will guide researchers in taking relevant measures to prevent and control American foulbrood. © 2023, The Author(s), under exclusive licence to Plant Science and Biodiversity Centre, Slovak Academy of Sciences (SAS), Institute of Zoology, Slovak Academy of Sciences (SAS), Institute of Molecular Biology, Slovak Academy of Sciences (SAS)

Antibacterial activity of bryophyte species against Paenibacillus larvae isolates

This study was performed to determine the antibacterial activity of methanol extracts of 23 bryophyte species against Paenibacillus larvae isolates that cause American foulbrood diseases in honeybee larvae. The honey and larva samples were collected from nine different locations of Rize in Turkey. A total of 22 gram-positive spore-forming bacteria were isolated from the larva and honey samples. According to the results of morphological, biochemical, and molecular (16S rRNA gene sequencing) tests, 10 isolates of the 22 gram-positive spore-forming bacteria were identified as P. larvae. A total of 10 bryophyte species (Polytrichum formasum,Polytrichum commune, Calliergonella cuspitada, Calliergonella lindbergi, Metzgeria conjugata, Isothecium alopecuroides, Syntrichia calcicola, Syntrichia intermedia, Tortella densa,and Grimmia alpestris) among 23 bryophytes showed good antimicrobial activity against P. larvae isolates according the results of agar-well diffusion method and minimal inhibition concentration experiments

Reversible hypertensive brainstem encephalopathy coexistent with acute intracerebral hemorrhage

We report two cases of reversible hypertensive brainstem encephalopathy (RHBE) syndrome with other concurrent manifestations of malignant hypertension. RHBE was accompanied by hypertensive putaminal hemorrhage in one case, and by intra-ventricular hemorrhage resulting acute obstructive hydrocephalus along with multiple small acute infarctions in the other. Diffusion-weighted imaging and corresponding apparent diffusion coefficient mapping disclosed increased diffusion suggestive of vasogenic edema centered in the pons. No lesion indicative for posterior reversible encephalopathy syndrome (PRES) was noted in the supratentorial regions. These cases indicate that mechanisms involved in development of RHBE and hypertensive PRES may have some differences, albeit both share main lines. These cases also underline that awareness and knowledge about relatively specific clinic-radiological syndrome caused by RHBE seem to be critical. Acute treatment and early prognostification are just only accurate with such a familiarity

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, bacterio-phage 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 conse-quence, 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. Bio-informatic 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 togetherRecep Tayyip Eerdogan University (RTEU) Scientific Research Projects Program RTEU-FBA-2017-80

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.RTEU Scientific Research Projects RTEU-FBA-2017-80

Prevalence and phylogenetic analysis of Ascosphaera apis (Maassen ex Claussen) LS Olive & Spiltoir (1955) isolates from honeybee colonies in Turkey

Ascosphaera apis (Maassen ex Claussen) Olive et Spiltoir is a causal agent of chalkbrood disease and is widely found in mainly honeybee colonies worldwide. Chalkbrood disease rarely causes colony death, but severe infections can lead to a decrease in honeybee populations and as a result, a decrease in honey production or beekeeping byproducts. Therefore, it is important to determine the distribution and prevalence of this disease agent in a certain region in terms of controlling of it. In this study, it was aimed to identify chalkbrood disease agents affecting honeybee colonies especially in beekeeping places in Turkey, to isolate and identify them using morphological and molecular methods, especially ITS gene sequencing. Morphologically, pure cultures of clinically suspected samples in terms of chalkbrood disease were created and microscopically evaluated. After that, genomic DNA isolation was performed from pure cultures to amplify ITS gene region and the resulting gene sequences were used in molecular identification and phylogenetic analysis. For this purpose, a total of 1.193 hives were randomly selected from 400 apiaries in 40 provinces throughout Turkey between 2018 and 2019. As a result, the presence of A. apis was detected in 7 hives in 2018 and 19 hives in 2019, as a result a total of 26 samples were found to be positive. Accordingly, the infection rate was determined as 2.18%. The obtained results are thought to be important in terms of determining the spread rate of A. apis and the rate of chalkbrood disease in Turkey

Molecular detection of Chlamydia abortus and investigation of oxidative stress parameters in sheep fetuses in the Black Sea Region of Türkiye

Background: The causative agent of Chlamydiosis, also called enzootic abortion of sheep, is an obligate intracellular Gram-negative bacterium called Chlamydia abortus. Purpose: The aim of this study is to determine the prevalence of Chlamydiosis and changes in oxidative stress levels in sheep abortions in the Black Sea Region. Method: PCR analysis made from aborted fetuses of 393 sheep from 8 provinces between 2019–2021; Presence of C. abortus detected in 65 aborted fetuses (16.5%). In the study, oxidative stress parameters such as CP, MDA, TOS, TAS and OSI levels were investigated. Result: CP, MDA, TOS, TAS and OSI values were respectively as 6.92 ± 2.29 mg/mL, 28.8 ± 4.79 µmol/g, 14.01 ± 7.25 µmol H2O2 Eq/L, 2.19 ± 0.67 mmol Trolox Eq/L, 6.94 ± 4.31 AU in the control group. In the chlamydia infected group, CP, MDA, TOS, TAS and OSI values were 10.96 ± 6.75 mg/mL, 92.31 ± 73.08 µmol/g, 36.04 ± 28.31 µmol H2O2 Eq/L, 1.91 ± 1.02 mmol Trolox Eq/L, 22.39 ± 19.5 AU was determined. Conclusions: CP, MDA, TOS and OSI levels were found to be significantly higher (P 0.05). As a result, it was determined that C. abortus was found at a substantial rate in small ruminant abortion cases in the Black Sea Region. In addition, it was determined for the first time in Türkiye that the oxidative stress increased in abortions caused by C. abortus and infected small ruminants could not produce enough antioxidants. It is thought that this result may be useful in the diagnosis of C. abortus

Antibacterial effects of anatolian propolis on paenibacillus larvae

Paenibacillus larvae (P. larvae) is a pathogenic bacterium causing American Foulbrood Disease (AFB) in honeybee larvae. It is necessary to develop alternative strategies for the control of this disease due to the serious honeybee colonies loses and the use of antibiotics. Recent studies are aimed at the investigating natural fighting agents against P. larvae. In our study, it was aimed to demonstrate potential antibacterial efficacy of ethanol extract of Anatolian Propolis (EAP) against P. larvae strains PB35 and SV35 which were isolated in Turkey. The total phenolic content (TPC) and flavonoid content (TFC) of EEAP were determined as 181.73±5.20 mg Gallic Acid Equivalents (GAE)/g, and 42.33±1.40 mg Quercetin Equivalents (QE)/g, respectively. It was found that EAP contains different amounts of ferulic, caffeic, coumaric acids, rutin, and caffeic acid phenethyl ester (CAPE). The antibacterial activity of the EAP was determined by using agar-well diffusion, microdilution, and Bioscreen C techniques. The Minimal Inhibition Concentration (MIC) values of the EAP were determined as 74.87 ?g/ml against strain PB35 and SV35 using both microdilution and Bioscreen C teqnique. In both techniques, Minimal Bactericidal Concentration (MBC) values of the EAP were evaluated as 149 and 598.4 ?g/ml against strain PB35 and SV35, respectively. The fact that EAP shows low concentrations of bacteriostatic (MIC) and bactericide (MBC) activity values against P. larvae strains, spore-forming bacilli, which are agents of AFB disease, suggests that it may be a potential source in AFB disease control. © The Author(s), 2021.Recep Tayyip Erdogan Üniversitesi, RTEU: RTEU-BAP 2015.53001.102.03.04Funding: This work was supported by the Recep Tayyip Erdoğan University research fund (RTEU-BAP 2015.53001.102.03.04. Thanks for their financial supports.Funding: This work was supported by the Recep Tayyip Erdo?an University research fund (RTEU-BAP 2015.53001.102.03.04. Thanks for their financial supports.2-s2.0-8511957887

Antibacterial activity of bryophyte species against Paenibacillus larvae isolates

This study was performed to determine the antibacterial activity of methanol extracts of 23 bryophyte species against Paenibacillus larvae isolates that cause American foulbrood diseases in honeybee larvae. The honey and larva samples were collected from nine different locations of Rize in Turkey. A total of 22 gram-positive spore-forming bacteria were isolated from the larva and honey samples. According to the results of morphological, biochemical, and molecular (16S rRNA gene sequencing) tests, 10 isolates of the 22 gram-positive spore-forming bacteria were identified as P. larvae. A total of 10 bryophyte species (Polytrichum formasum,Polytrichum commune, Calliergonella cuspitada, Calliergonella lindbergi, Metzgeria conjugata, Isothecium alopecuroides, Syntrichia calcicola, Syntrichia intermedia, Tortella densa,and Grimmia alpestris) among 23 bryophytes showed good antimicrobial activity against P. larvae isolates according the results of agar-well diffusion method and minimal inhibition concentration experiments.This study was performed to determine the antibacterial activity of methanol extracts of 23 bryophyte species against Paenibacillus larvae isolates that cause American foulbrood diseases in honeybee larvae. The honey and larva samples were collected from nine different locations of Rize in Turkey. A total of 22 gram-positive spore-forming bacteria were isolated from the larva and honey samples. According to the results of morphological, biochemical, and molecular (16S rRNA gene sequencing) tests, 10 isolates of the 22 gram-positive spore-forming bacteria were identified as P. larvae. A total of 10 bryophyte species (Polytrichum formasum,Polytrichum commune, Calliergonella cuspitada, Calliergonella lindbergi, Metzgeria conjugata, Isothecium alopecuroides, Syntrichia calcicola, Syntrichia intermedia, Tortella densa,and Grimmia alpestris) among 23 bryophytes showed good antimicrobial activity against P. larvae isolates according the results of agar-well diffusion method and minimal inhibition concentration experiments