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Molecular epidemiology and phylogenetic analysis of Anaplasma ovis and Anaplasma marginale in Ixodidae infesting livestock in northwestern Iran.

BACKGROUND: Anaplasma marginale and Anaplasma ovis are tick-borne obligate intracellular bacteria causing anaplasmosis in cattle and small ruminants, respectively, with considerable economic losses worldwide. Given the favorable ecological conditions for tick survival and the limitation of data on Anaplasma spp. in local tick populations in northwestern Iran, this study aimed to molecular and phylogenetic analysis of A. ovis and A. marginale in Ixodidae infesting livestock in northwestern Iran. METHODS: In this cross-sectional study, a total of 780 ixodid ticks were collected from livestock across 198 herds in 11 counties of Ardabil Province during 2025. Ticks were morphologically identified and grouped into pools based on species and host type. Genomic DNA was extracted using a commercial kit and molecular detection of A. ovis and A. marginale was performed using PCR assays targeting the 16&#xa0;S rRNA and groEL genes. Positive samples were submitted for Sanger sequencing to confirm the identity of Anaplasma spp., phylogenetic analysis was conducted using reference sequences from GenBank (NCBI) using MEGA software (version 12). Statistical analyses were conducted using SPSS (version 25), and associations between categorical variables were assessed using Fisher's exact test (p < 0.05). RESULTS: Eight tick species belonging to three genera were identified. Among the hosts, sheep exhibited the highest infestation rate (49.3%). Hyalomma anatolicum anatolicum was the most prevalent species (25.3%) and was present in all sampled counties. Tick distribution varied significantly among host species (p = 0.003) and geographic locations (p = 0.002). PCR analysis detected A. ovis DNA in 16.6% (4/24) and A. marginale DNA in 8.3% (2/24) of tick pools. Positive pools were primarily associated with Rhipicephalus spp. and Dermacentor marginatus. According to the results of the statistical analysis a significant association was found between tick species and host type (&#x3c7;&#xb2; = 13.87, p = 0.0031), with Hyalomma anatolicum anatolicum more prevalent in sheep (p = 0.001). Tick abundance varied across counties (&#x3c7;&#xb2; = 16.42, p = 0.002), with highest densities in Nir, Khalkhal, and Kowsar (&#x3c7;&#xb2; = 14.21, p = 0.0028). No significant association was observed between Anaplasma positivity and tick species (p = 0.21) or host type (p = 0.09). CONCLUSIONS: The detection of A. ovis and A. marginale DNA in ixodid tick pools indicates their circulation in Ardabil Province. However, due to pooled sampling and the limited number of positive samples, the infection rate at the individual tick level could not be determined. These findings also highlight the importance of a One Health approach, considering the interconnected roles of animal health, tick vectors, and the environment in the transmission and control of tick-borne diseases.

Animals↗

Phylogenetic analysis of rubella virus including new genotype I isolates.

Infection during the first trimester of gestation with rubella virus (RV) is highly teratogenic. Embryopathy is a frequent outcome of the primary natural infection with wild type RV during pregnancy while accidental immunisation with life attenuated vaccine has apparently little or no adverse effect. Although the nucleic acid sequence of RV is exceptionally stable, differences between the vaccine and wild type viruses could play a role in the pathogenesis of intrauterine RV infection. Phylogenetic analysis of eight complete sequences (including two new isolates described in this paper, three vaccine strains, three cell culture adapted wild type viruses) confirms a striking low divergence of the RV genome. A variable region (amino acid residues 697-800) within the gene coding for the nonstructural protein NSP1 was defined. Phylogenetic analysis revealed a strong positive selection in this region. Multiple passages in vivo or in vitro did not account for this variability. As the function of the variable region has not yet been elucidated, reasons for and significance of positive selection are still speculative. It is conceivable that the variable region in NSP1 contributes to the molecular basis of RV embryopathy and other complications of postnatal RV infection.

Animals↗

Phylogenetic analysis of enteric species of the family Enterobacteriaceae using the oriC-locus.

Phylogenetic analysis of 38 enteric species belonging to the Enterobacteraceae family was carried out using the non-coding locus oriC, the chromosomal replication origin. The oriC loci were amplified with conserved oligonucleotides and the PCR fragments were sequenced directly. The results establish a phylogenetic tree for the classification of different species of the genera Escherichia, Shigella, Salmonella, Enterobacter, Citrobacter, Klebsiella, Raoultella, Kluyvera, Cedecea and Buttiauxella. Functional important protein-binding sites located in oriC are well conserved throughout the enteric group. More over, due to a high overall divergence value phylogenetic trees were robust and well supported by bootstrap analysis. In comparison with 16S rDNA analysis, the oriC sequences indicated a greater evolutionary divergence for bacteria. We propose that the oriC locus might be a suitable phylogenetic marker for the identification and classification of bacteria, in particular for closely related species.

Bacterial Proteins↗

Complete genome sequence and phylogenetic analysis of hepatitis B virus isolated from Turkish patients with chronic HBV infection.

Hepatitis viruses are the leading causes of chronic liver disease resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma in the world and also in Turkey. Although Turkey has an intermediate rate of hepatitis B virus (HBV) infection with a prevalence reported as 5%, a complete HBV genome sequence has not been published. In this study, the molecular characterization and phylogenetic analysis are described of 11 complete HBV genomes isolated from 11 naïve patients (5 male, 6 female; ages: 18--54 years old, median 35 years old) with chronic HBV infection. Of 11 patients, 7 and 4 were HBeAg positive/anti-HBe negative and HBeAg negative/anti-HBe positive, respectively. All patients had no co-infection with HCV, HDV, or HIV. HBV DNA was extracted from the sera of the patients. The complete genome was amplified by PCR and cloned into a TA vector. The PCR products were sequenced directly and the complete HBV genome sequences were determined. Ten HBV genomes were 3182 base pairs in length. There was a 183 bp deletion (between nucleotides 2987--3169) in pre-S region in one HBeAg positive patient. There were two pre-core stop codons (G1896A) in two HBeAg negative and three core promoter dual mutations (T1762/A1764) in one HBeAg positive and two HBeAg negative patients' HBV genomes. Phylogenetic analysis of all complete genomes yielded that all Turkish sequences were clustered in genotype D branch (ten in subgenotype D1 and one in subgenotype D2). The analysis of S gene amino acid sequences revealed that surface gene subtypes of one and ten HBV strains were subtype ayw3 and ayw2, respectively. This study indicates that Turkish patients with chronic hepatitis B infection show very little genotypic heterogeneity. Genotype D of HBV DNA and subtype ayw2 of surface gene represent almost the whole Turkish patient population infected with HBV.

Adult↗

Sequence determination and phylogenetic analysis of the Akabane bunyavirus S RNA genome segment.

The nucleotide sequence of the small (S) RNA segment of Akabane (AKA) bunyavirus was determined. The segment is 858 nucleotides long and contains two overlapping open reading frames (ORFs), which encode the nucleocapsid (N) and nonstructural (NSs) proteins, consistent with other bunyaviruses. Comparisons with the Aino virus S RNA sequence indicated that there is 73.5% identity in nucleotide sequence. However, the sequence identity of the 5' non-coding region of the genomic RNA between these two viruses is only 55%. The N ORFs from 20 Japanese and 2 Australian isolates of AKA virus were sequenced and subjected to phylogenetic analysis. This suggested that AKA virus has evolved in multiple lineages. Twenty-three isolates were grouped into three major clusters, and the cluster which includes recent isolates was subdivided into two branches. Thus, phylogenetic analysis of the AKA virus N protein gene gives a greater insight into bunyavirus evolution.

Genome, Viral↗

Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu.

Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions.

Amino Acid Sequence↗

Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions.

Porcine circovirus type 2 (PCV2) has been associated with a newly identified and described disease in swine called the postweaning multisystemic wasting syndrome (PMWS). An association of PCV2 with various other clinical conditions in pigs are also increasingly being reported. To date molecular studies to determine the extent of genetic variations of this virus have been limited. To more fully understand the extent of genetic diversity we report the sequencing of 34 PCV2 strains from Eastern Canada originating from a variety of clinical conditions spanning years 1990-2001 along with the phylogenetic analysis of these strains and that of 36 PCV2 sequences published in GenBank. Sequence analysis of the complete genome indicated that the Canadian PCV2 strains analyzed in the present study are closely related to each other but also to other PCV2 strains originating from Western Canada, US, Europe and Asia. Sequence analysis of ORF1 and ORF2 genes of the 34 strains revealed that the extent of nucleotide variation was greater for the ORF2 than for the ORF1. The amino acid sequences alignment of the PCV2 capsid protein identified three major regions of amino acid heterogeneity, two of which correspond with dominant immunoreactive areas. No repeatable amino acid motifs for these two regions could be associated with PCV2 strains identified from cases of PMWS or other clinical conditions. Phylogenetic analysis of all 70 strains revealed one large cluster composed of strains from Europe, Taiwan, China and Canada. This large cluster could be divided in several sub clusters, in two of which Canadian strains were found closely related to strains from Germany. The remaining strains from Canada and the US were spread in small groupings along the phylogenetic tree and an association with geographic origin could not be established. The genomic characterizations performed in this study indicate that PCV2 strains associated with PMWS are scattered throughout the phylogenetic tree often in groupings including PCV2 strains identified from cases other that PMWS such as, porcine reproductive and respiratory syndrome (PRRS), generalized tremors, porcine dermatitis and nephropathy syndrome (PDNS), arthritis, nervous signs, erysipelas and even healthy pigs.

Amino Acid Sequence↗

Identification and phylogenetic analysis of 15 MHC class II DRB1 beta1 expressed alleles in a ewe-lamb flock.

Ovar-DRB1 is part of the major histocompatibility complex (MHC) class II of sheep and functions by presenting extracellular-derived peptides to the immune system. Although there are over 100 different Ovar-DRB1 DNA sequences reported in GenBank, only two Ovar-DRB1 mRNA sequences have been reported. As a first step in understanding MHC Class II function as it relates to disease progression in sheep, Ovar-DRB1 transcripts encoding the peptide-binding site or the first domain (beta1) of Ovar-DRB1 in a 32-ewe-lamb flock were identified and characterized by using reverse transcriptase-polymerase chain reaction, cloning, sequencing, and phylogenetic analysis. Fourteen new Ovar-DRB1 beta1 cDNA sequences out of a total of 15 Ovar-DRB1 beta1 cDNA sequences in a ewe-lamb flock of 32 sheep were identified. One Ovar-DRB1 beta1 cDNA sequence was 100% identical to M93432, one of the two Ovar-DRB1 mRNA sequences reported in GenBank. Twelve out of 15 Ovar-DRB1 beta1 cDNA sequences were 100% identical to the corresponding previously reported Ovar-DRB1 genomic DNA sequences, indicating that these Ovar-DRB1 genomic DNA sequences are also transcribed. One of three of the remaining Ovar-DRB1 beta1 cDNA sequences, DRB1*07012, had a synonymous substitution resulting in an identical deduced amino acid sequence to DRB1*0701. Two of the remaining three Ovar-DRB1 beta1 cDNA sequences had nucleotide differences and subsequent deduced amino acid sequence differences when compared to known Ovar-DRB1 beta1 genomic DNA sequences, and therefore, DRB1*0206 and DRB1*0353 represent new Ovar-DRB1 beta1 expressed alleles. Phylogenetic analysis of the 15 Ovar-DRB1 beta1 cDNA sequences revealed that DRB1*0206 had a strong phylogenetic relationship to DRB1*0203, and DRB1*0353 had a strong phylogenetic relationship to DRB1*0303.

Alleles↗

Phylogenetic analysis of Streptomyces spp. isolated from potato scab lesions in Korea on the basis of 16S rRNA gene and 16S-23S rDNA internally transcribed spacer sequences.

The 16S rRNA gene sequences for 34 strains, including 11 isolates, were determined to classify scab-causing Streptomyces spp. and relatives isolated from potato scab lesions collected in Jeju, Korea. The 16S-23S rDNA internally transcribed spacer (ITS) sequences were determined to investigate whether the 16S-23S ITS region is useful for analysing intra- and interspecific relationships in these bacteria. On the basis of phylogenetic analysis of 16S rRNA gene sequences, most of the isolates were classified as Streptomyces scabiei and Streptomyces acidiscabies. Isolate KJO61 was placed in an ambiguous taxonomic position between Streptomyces reticuliscabiei and Streptomyces turgidiscabies. 16S-23S ITS region sequence analysis showed that tRNA genes were not found in this region of Streptomyces spp. The 16S-23S ITS regions of Streptomyces spp. exhibited various lengths and highly variable sequence similarities (35-100%) within strains as well as intra- and interspecies. It was revealed that Streptomyces europaeiscabiei could be clearly differentiated from Streptomyces scabiei. However, it was clarified that ITS regions are not useful in phylogenetic analysis of Streptomyces spp.

Base Sequence↗

QAlign: quality-based multiple alignments with dynamic phylogenetic analysis.

Integrating different alignment strategies, a layout editor and tools deriving phylogenetic trees in a 'multiple alignment environment' helps to investigate and enhance results of multiple sequence alignment by hand. QAlign combines algorithms for fast progressive and accurate simultaneous multiple alignment with a versatile editor and a dynamic phylogenetic analysis in a convenient graphical user interface.

Algorithms↗

[Possible differentiation of mitotypes in mixed biological subjects based on phylogenetic analysis of mitochondrial DNA].

A possibility was investigated to use the phylogenetic analysis of mitochondrial (mt) DNA to differentiate between the mito-types in mixed subjects in forensic medical examinations. The suggested methodological approach is based on the use of electrophoregram and on the comparison of such data with the information about phylogenic individual (mt) DNA lines.

Base Sequence↗

Identification, characteristic and phylogenetic analysis of type II DNA topoisomerase gene in Giardia lamblia.

The genes encoding type II DNA topoisomerases were investigated in Giardia lamblia genome, and a type IIA gene, GlTop 2 was identified. It is a single copy gene with a 4476 bp long ORF without intron. The deduced amino acid sequence shows strong homology to eukaryotic DNA Top 2. However, some distortions were found, such as six insertions in the ATPase domain and the central domain, a approximately 100 aa longer central domain; a approximately 200 aa shorter C-terminal domain containing rich charged residues. These features revealed by comparing with Top 2 of the host, human, might be helpful in exploiting drug selectivity for antigiardial therapy. Phylogenetic analysis of eukaryotic enzymes showed that kinetoplastids, plants, fungi, and animals were monophyletic groups, and the animal and fungi lineages shared a more recent common ancestor than either did with the plant lineage; microsporidia grouped with fungi. However, unlike many previous phylogenetic analyses, the "amitochondriate"G. lamblia was not the earliest branch but diverged after mitochondriate kinetoplastids in our trees. Both the finding of typical eukaryotic type IIA topoisomerase and the phylogenetic analysis suggest G. lamblia is not possibly as primitive as was regarded before and might diverge after the acquisition of mitochondria. This is consistent with the recent discovery of mitochondrial remnant organelles in G. lamblia.

Animals↗

Identification and prevalence of Ehrlichia chaffeensis infection in Haemaphysalis longicornis ticks from Korea by PCR, sequencing and phylogenetic analysis based on 16S rRNA gene.

Genomic DNAs extracted from 1,288 Haemaphysalis longicornis ticks collected from grass vegetation and various animals from nine provinces of Korea were subjected to screening by genus-specific (Ehrlichia spp. or Anaplasma spp.) real-time TaqMan PCR and speciesspecific (E. chaffeensis) nested-PCR based on amplification of 16S rRNA gene fragments. In all, 611 (47.4%) ticks tested positive for genus-specific amplification of 116 bp fragment of 16S rRNA of Ehrlichia spp. or Anaplasma spp. Subsequently, 396 bp E. chaffeensis-specific fragment of 16S rRNA was amplified from 4.2% (26/611) tick samples. The comparison of the nucleotide sequence of 16S rRNA gene from one tick (EC-PGHL, GeneBank accession number AY35042) with the sequences of 20 E. chaffeensis strains available in the database showed that EC-PGHL was 100% identical or similar to the Arkansas (AF416764), the Sapulpa (U60476) and the 91HE17 (U23503) strains. The phylogenetic analysis also revealed that the E. chaffeensis EC-PGHL formed a single cluster with the above strains. This is the first study to report molecular detection and phylogenetic analysis of E. chaffeensis from H. longicornis ticks in Korea. The implicit significance of E. chaffeensis infection in H. longicornis ticks in Korea is discussed.

Anaplasma↗

Quantification of homoplasy for nucleotide transitions and transversions and a reexamination of assumptions in weighted phylogenetic analysis.

Nucleotide transitions are frequently down-weighted relative to transversions in phylogenetic analysis. This is based on the assumption that transitions, by virtue of their greater evolutionary rate, exhibit relatively more homoplasy and are therefore less reliable phylogenetic characters. Relative amounts of homoplastic and consistent transition and transversion changes in mitochondrial protein coding genes were determined from character-state reconstructions on a highly corroborated phylogeny of mammals. We found that although homoplasy was related to evolutionary rates and was greater for transitions, the absolute number of consistent transitions greatly exceeded the number of consistent transversions. Consequently, transitions provided substantially more useful phylogenetic information than transversions. These results suggest that down-weighting transitions may be unwarranted in many cases. This conclusion was supported by the fact that a range of transition: transversion weighting schemes applied to various mitochondrial genes and genomic partitions rarely provided improvement in phylogenetic estimates relative to equal weighting, and in some cases weighting transitions more heavily than transversions was most effective.

Base Sequence↗

Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members.

Cadherins play an important role in specific cell-cell adhesion events. Their expression appears to be tightly regulated during development and each tissue or cell type shows a characteristic pattern of cadherin molecules. Inappropriate regulation of their expression levels or functionality has been observed in human malignancies, in many cases leading to aggravated cancer cell invasion and metastasis. The cadherins form a superfamily with at least six subfamilies, which can be distinguished on the basis of protein domain composition, genomic structure, and phylogenetic analysis of the protein sequences. These subfamilies comprise classical or type-I cadherins, atypical or type-II cadherins, desmocollins, desmogleins, protocadherins and Flamingo cadherins. In addition, several cadherins clearly occupy isolated positions in the cadherin superfamily (cadherin-13, -15, -16, -17, Dachsous, RET, FAT, MEGF1 and most invertebrate cadherins). We suggest a different evolutionary origin of the protocadherin and Flamingo cadherin genes versus the genes encoding desmogleins, desmocollins, classical cadherins, and atypical cadherins. The present phylogenetic analysis may accelerate the functional investigation of the whole cadherin superfamily by allowing focused research of prototype cadherins within each subfamily.

Amino Acid Sequence↗

Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis.

Histone deacetylases (HDACs) modify core histones and participate in large regulatory complexes that both suppress and enhance transcription. Recent studies indicate that some HDACs can act on non-histone proteins as well. Interest in these enzymes is growing because HDAC inhibitors appear to be promising therapeutic agents against cancer and a variety of other diseases. Thus far, 11 members of the HDAC family have been identified in humans, but few have been characterized in detail. To better define the biological function of these proteins, make maximal use of studies performed in other systems, and assist in drug development efforts, we have performed a phylogenetic analysis of all HDAC-related proteins in all fully sequenced free-living organisms. Previous analyses have divided non-sirtuin HDACs into two groups, classes 1 and 2. We find that HDACs can be divided into three equally distinct groups: class 1, class 2, and a third class consisting of proteins related to the recently identified human HDAC11 gene. We term this novel group "class 4" to distinguish it from the unrelated "class 3" sirtuin deacetylases. Analysis of gene duplication events indicates that the common ancestor of metazoan organisms contained two class 1, two class 2, and a single class 4 HDAC. Examination of HDAC characteristics in light of these evolutionary relationships leads to functional predictions, among them that self-association is common among HDAC proteins. All three HDAC classes (including class 4) exist in eubacteria. Phylogenetic analysis of bacterial HDAC relatives suggests that all three HDAC classes precede the evolution of histone proteins and raises the possibility that the primary activity of some "histone deacetylase" enzymes is directed against non-histone substrates.

Eukaryotic Cells↗

Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins.

BACKGROUND: The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs. RESULTS: The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis. CONCLUSIONS: The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.

Amino Acid Sequence↗

Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of the gyrB gene sequence.

Phylogenetic analysis of about 200 strains of Salmonella, Shigella, and Escherichia coli was carried out using the nucleotide sequence of the gene for DNA gyrase B (gyrB), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of Salmonella, Shigella, and Escherichia coli in which Salmonella forms a cluster separate from but closely related to Shigella and E. coli. In comparison with 16S rRNA analysis, the gyrB sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the gyrB gene might be a useful tool for differentiating between closely related species of bacteria such as Shigella spp. and E. coli. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable gyrB sequence region can identify unknown bacteria to the species level. In summary, we have shown that gyrB sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.

DNA Gyrase↗