Base Pair Fragment Research Articles

Redescription of Manayunkia caspica Annenkova, 1929 and M. danubialis Băcescu, 1944 n. comb. (Fabriciidae, Sabellida, Polychaeta).

The spread of the subspecies M. caspica danubialis Băcescu, 1944, which has been found in the Danube since 1944, into the upper section of the Danube as far as Austria was the reason for describing the characters of this species and comparing it with the nominotypical subspecies M. caspica caspica Annenkova, 1929, which lives in the Caspian Sea. Both subspecies are similar in the most important diagnostic characters, such as the number of branches of the radioles and the presence and number of pseudospatulate chaetae on thoracic chaetigers. However, they differ in other, less conspicuous characters, such as the number of thoracic uncini and the length of chaetigers. But these characters vary depending on the size of the specimens. Both subspecies inhabit completely different habitats. Based on the ecological species concept, the spatially and ecologically separated populations are regarded as distinct species, M. caspica, and M. danubialis n. comb. The occurrence of transitional uncini in both species and the presence of a brood chamber in the females of M. caspica are described for the first time. The structure of the radiolar crown is also described for both species. A molecular barcode for M. danubialis n. comb., a 654 base pair fragment of cytochrome c oxidase I is presented, allowing a fast and simple identification of this species by using a robust PCR/sequencing approach.

Molecular and morphometric study of Brazilian populations of Psychodopygus davisi.

In this study, we analysed the molecular and morphometric differences of several populations of the putative sand fly vector Psychodopygus davisi (Root, 1934) (Diptera, Psychodidae, Phlebotominae) in Brazil. We amplified the 658 base pair fragments of the DNA barcoding region-cytochrome c oxidase subunit 1 (COI) gene-for 57 specimens of P. davisi and three specimens of Psychodopygus claustrei (Abonnenc, Léger & Fauran, 1979). We merged our data with public sequences of the same species available from GenBank. Then, the combined dataset-87 sequences and 20 localities-was analysed using population structure analysis and different species delimitation approaches. Geometric morphometry of wings was performed for 155 specimens of P. davisi populations from the North, Midwest and Southeast Brazilian regions, analysing the differences in centroid sizes and canonical variates. Molecular analysis indicated high intraspecific genetic distance values for P. davisi (maximum p distance = 5.52%). All algorithms identified P. davisi and P. claustrei as distinct molecular taxonomic units, despite the low interspecific distance (p distance to the nearest neighbour = 4.79%). P. davisi sequences were split into four genetic clusters by population structure analysis and at least five genetic lineages using intermediate scenarios of the species delimitation algorithms. The species validation analysis of BPP strongly supported the five-species model in our dataset. We found high genetic diversity in this taxon, which is in agreement with its wide geographic distribution in Brazil. Furthermore, the wing analysis showed that specimens from the Southeast Region of Brazil are different from those in the North and the Midwest. The evolutionary patterns of P. davisi populations in Brazil suggest the presence of candidate species, which need to be validated in future studies using a more comprehensive approach with both genomic data and morphological characters.

Identification of Intron Retention in the Slc16a3 Gene Transcript Encoding the Transporter MCT4 in the Brain of Aged and Alzheimer-Disease Model (APPswePS1dE9) Mice.

The monocarboxylate transporter 4 (MCT4; Slc16a3) is expressed in the central nervous system, notably by astrocytes. It is implicated in lactate release and the regulation of glycolytic flux. Whether its expression varies during normal and/or pathological aging is unclear. As the presence of its mature transcript in the brain of young and old mice was determined, an unexpectedly longer RT-PCR fragment was detected in the mouse frontal cortex and hippocampus at 12 vs. 3 months of age. Cultured astrocytes expressed the expected 516 base pair (bp) fragment but treatment with IL-1β to mimic inflammation as can occur during aging led to the additional expression of a 928 bp fragment like that seen in aged mice. In contrast, cultured pericytes (a component of the blood-brain barrier) only exhibited the 516 bp fragment. Intriguingly, cultured endothelial cells constitutively expressed both fragments. When RT-PCR was performed on brain subregions of an Alzheimer mouse model (APPswePS1dE9), no fragment was detected at 3 months, while only the 928 bp fragment was present at 12 months. Sequencing of MCT4 RT-PCR products revealed the presence of a remaining intron between exon 2 and 3, giving rise to the longer fragment detected by RT-PCR. These results unravel the existence of intron retention for the MCT4 gene in the central nervous system. Such alternative splicing appears to increase with age in the brain and might be prominent in neurodegenerative diseases such as Alzheimer's disease. Hence, further studies in vitro and in vivo of intron 2 retention in the Slc16a3 gene transcript are required for adequate characterization concerning the biological roles of Slc16a3 isoforms in the context of aging and Alzheimer's disease pathology.

DNA Barcoding Culicoides Biting Midges (Diptera: Ceratopogonidae) in Northeast Brazil.

Biting midges of the genus Culicoides are small insects associated with the transmission of several pathogens, which requires the correct identification of the species, for implementation of effective strategies against these insects. However, many species are difficult to identify only by morphological characters. Therefore, the use of molecular methods can help in the taxonomy and systematics of this group. Here, the DNA barcode approach was evaluated for nine species of Culicoides from the State of Maranhão, Brazil. We generated 39 sequences from a 476 bp (base pairs) fragment of the cytochrome c oxidase subunit I (COI) mitochondrial gene. To assess the usefulness of COI barcodes for the identification of these species, paired genetic distances from intra and interspecific comparisons and phylogenetic trees were generated in MEGA and RAxML/BEAST softwares, respectively. In addition, species delimitation was performed using the PTP, GMYC, and ABGD algorithms. The intra and interspecific genetic distances showed a clear distinction between them, demonstrating that, for the taxa studied, there can hardly be ambiguous identifications with barcodes. In the same sense, the phylogenetic reconstruction resulted in well-supported clades for all morphospecies analyzed.

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

DNA contamination and taxonomic diversity hinder eDNA research on freshwater stingray species: Comment on Lim & Then (2022)

Recently, Lim & Then (2022; Endang Species Res 48:43-50) used an environmental DNA (eDNA) PCR-based method for detecting the presence of an endangered species of freshwater stingray, Fluvitrygon kittipongi (Dasyatidae), in the Pahang River basin, east Peninsular Malaysia. For that, they designed a species-specific pair of primers to amplify a 196 base pair (bp) fragment of the cytochrome c oxidase I (COI) gene. Lim & Then (2022) reported positive detection of this species along an upstream stretch of the Pahang River during the inter-monsoon and the southwest monsoon seasons, with the PCR-amplified eDNA sequences identical to the reference COI sequences of F. kittipongi collected from the Perak River basin, west Peninsular Malaysia. Here, we argue that such positive results are likely the consequence of a DNA contamination because the 196 bp COI fragments of F. kittipongi from the Pahang River and the Perak River differ from each other by 4 nucleotide substitutions. The source of contamination in Lim & Then (2022) could have been the Perak samples the authors handled to develop the primers. We also briefly discuss the potential impact of the presumed co-occurrence of freshwater stingrays of the genus Fluvitrygon in the Pahang River on the specificity of the F. kittipongi-specific primers designed by Lim & Then (2022).

Report of two distinct ribotypes in ITS sequences of Phalarisarundinacea (Poaceae) in western Canada and Alaska.

Phalarisarundinacea L. (reed canary grass) is a widely occurring grass throughout the Northern Hemisphere. In North America, it is thought to consist of introduced agricultural forms from Europe as well as native populations. During a survey of Phalarisarundinacea in western Canada, we discovered two distinct ribotypes in the sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA: one full length (ITS-long) and one with a seven base pair deletion (ITS-short). In addition, ITS-long plants have fixed heterozygosity indicating possible polyploidy. Phylogenetic analysis reveals that ITS-short is a unique ribotype that characterises an intraspecific clade. We designed an efficient PCR-based assay that allows sizing of a 238/245 base pair fragment in a capillary sequencer. This approach provides a novel marker that could be useful in future surveys of Phalarisarundinacea.

A Contribution to the Systematics of Sunda Shelf Angle-Headed Dragons (Agamidae: Gonocephalus) with the Description of New Taxa from Sumatra

We review morphology of Sunda Shelf Gonocephalus with an emphasis on Sumatran and Javan species. At least 15 species and subspecies inhabit Peninsular Malaysia, Sumatra, Java, Borneo, and adjacent smaller continental islands. Following analysis of external morphology, we provide a dichotomous key to Sunda Shelf Gonocephalus and resolve two taxonomic problems with this group of lizards. Three populations of Gonocephalus doriae on Borneo, Peninsular Malaysia, and Sumatra are recognized as subspecies, because they lack concordance of multiple morphological differences and have low genetic divergence in a 556 base-pair fragment of the 16S rRNA gene. Described as new herein, G. doriae brevis from Aceh and North Sumatra differs from G. d. abbotti and G. d. doriae in usually having more scales around midbody and a relatively shorter tail with fewer dark bands. Though previously reported as lost, a syntype of G. d. doriae (MSNG 29152) is designated as lectotype, illustrated, and described. Also described as new, G. inauris from high elevations of the Bukit Barisan Range of Bengkulu, Sumatra, is a species of the G. megalepis Group differing from all congeners in having 7/6 (vs. 8–19) loreals separating the last canthal and supralabials, 8/8 (11–27) infraorbitals, and 58 (73–153) scales around midbody. In this new species, distinctly enlarged suboculars broadly contact its supralabials, whereas 1–4 lorilabials separate the suboculars and supralabials in congeners. High genetic divergence in the new species mirrors its high level of morphological divergence.

Variation in Pyrethroid Resistance Phenotypes in Anopheles darlingi in an Area with Residual Malaria Transmission: Warning of Suspected Resistance in French Guiana.

Anopheles darlingi is the main vector of malaria in South America. In French Guiana, malaria transmission occurs inland and along the rivers with a regular reemergence in the lower Oyapock area. Control against malaria vectors includes indoor residual spraying of deltamethrin and the distribution of long-lasting impregnated bednets. In this context, the level of resistance to pyrethroids was monitored for 4 years using CDC bottle tests in An. darlingi populations. A loss of susceptibility to pyrethroids was recorded with 30-minute knock-down measured as low as 81%. However, no pyrethroid molecular resistance was found by sequencing a 170 base pair fragment of the S6 segment of domain II of the voltage-gated sodium channel gene. Fluctuation of resistance phenotypes may be influenced by the reintroduction of susceptible alleles from sylvatic populations or by other mechanisms of metabolic resistance.

Simultaneous molecular detection of Anaplasma marginale and Theileria annulata in cattle blood samples collected from Pakistan-Afghanistan boarder region.

Theileria annulata (T. annulata) and Anaplasma marginale (A. marginale) are among the most extensively reported tick borne pathogens and are associated with huge economic losses worldwide. A total of 298 cattle blood samples were screened to report the presence of these two pathogens. The samples were collected from apparently healthy cattle (Achai, n = 155, Jersy, n = 88 and crossbred, n = 55) in Bajaur district of Khyber Pakhtunkhwa (KPK) during June and July of 2022. A total of 31 out of 298 cattle (10.4%) were found infected with T. annulata as PCR amplified a 156 base pair fragment from Tams-1 gene of T. annulata from their blood. While 16/298 animals (5.4%) were found infected with A. marginale as they amplified a 382 base pair fragment specific for msp5 gene of this bacterium. Three animals (1%) were found co infected. Cattle susceptibility to T. annulata infection was significantly higher than A. marginale infection (P < 0.001). Phylogenetic analysis revealed that Pakistani isolates of both detected pathogen clustered together and were closely related isolates from worldwide countries. Prevalence of T. annulata varied significantly among the sampling sites (P = 0.05) while no such association was observed for A. marginale among the tested cattle. Epidemiological data analysis revealed that none of the studied risk factors was found associated either with the prevalence of T. annulata or A. marginale (P > 0.05) among enrolled cattle. In conclusion, our study has revealed a relatively higher prevalence of T. annulata than A. marginale in cattle from the Bajaur district in KPK. This information is important for improving the productivity of the livestock sector, which is one of the main sources of income in the country. It is recommended that this data be taken into account for the development and implementation of effective tick control programs, as well as for the improvement of livestock management practices to prevent and manage TBDs in Pakistan.

Cryptic Lineage and Genetic Structure of Acanthopagrus pacificus Populations in a Natural World Heritage Site Revealed by Population Genetic Analysis

Recent studies have revealed extensive genetic differentiation among some populations of marine taxa that were previously believed to be essentially homogeneous because larvae are widely dispersed in ocean currents. Acanthopagrus pacificus is a commercially and ecologically important teleost fish that is endemic to shallow coastal waters and estuaries of some tropical and sub-tropical areas in the West Pacific Ocean. Here, we examined genetic structure and the inferred demographic history of A. pacificus populations from mtDNA control region sequence data. A 677–678 base-pair fragment was sequenced from 159 individuals sampled at three localities across the West Pacific Ocean. Haplotype diversity was high, ranging from 0.915 to 0.989, while nucleotide diversity was low to medium, ranging from 0.8% to 2.60%. Analysis of molecular variance (AMOVA) showed significant genetic subdivision (FST = 0.155, p &lt; 0.05) among sampled populations while pairwise FST estimates also revealed strong genetic differentiation among populations indicating that gene flow was restricted. Two distinct cryptic lineages were identified that were estimated to have diverged during the Pleistocene. In summary, contemporary factors including regional oceanic currents and self-recruitment are considered to have played significant roles in producing the population structure in this fish. In particular, the genetic information generated in the current study will allow appropriate fisheries management and conservation strategies to be developed for this important local fish in the waters around Iriomotejima Island, a World Heritage site.

Blood in the water: DNA barcoding of traded shark fins in Singapore

Overfishing has significantly decreased global shark populations, with some species experiencing reductions of approximately 70% over the last 50 years. Singapore is a major shark fin transhipment hub that helps to satisfy the global demand for shark fins, which are considered status symbols and reputed to have medicinal value in Asian culture. Despite the recognised and urgent need to better protect shark populations, the success of such efforts has been limited by the difficulties associated with visually identifying the species of shark from which the fins originated. In this study, we collected 451 shark fin tissue samples from a variety of local retail markets in Singapore. Using DNA barcoding techniques, we amplified a 350 base pair fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene from each to identify the species sold in Singapore. We identified 22 shark species, of which 17 are categorised as Threatened (Critically Endangered, Endangered or Vulnerable) under the IUCN Red List. Six of these species are also listed on Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

Pathology and Characterization of Fowlpox Virus Infection in a Turkey-Chicken Backyard Flock, Nigeria

Cutaneous scabs were seen on the nares and shanks in three grower turkeys in a mixed turkey-chicken flock in Langtang-north LGA of Plateau State, Nigeria. A chicken in the flock had diphtheritic membrane covering the mucous membranes of the oro-pharynx and the turkeys had cutaneous pox lesions on the nares and shank. Fowlpox virus infection was diagnosed by gross and histopathology and confirmed by isolation of the virus in chorioallantoic membrane of 9-12 weeks chicken embryonating eggs. The DNA of Fowl Pox Virus (FPV) was detected in the cutaneous scabs of the turkey and chicken using Polymerase Chain Reaction (PCR) which amplified a 578 base pairs fragment of the 4b core protein gene. Sequencing and phylogenetic analysis revealed that the fowlpox virus responsible for this infection clustered with the sublineage A1 genotype of clade A of fowlpox virus and shares 98% homology with the vaccine strain produced in Nigeria. The sequence has been deposited in the GenBank under the accession number MK435242. Our results confirmed the presence of FPV in the turkeys and suggest a predisposition by the chickens. We therefore suggest vaccination for small holder poultry to mitigate against mortality in turkeys and chickens and molecular epidemiology of fowlpox viruses in Nigeria to unravel viral evolution.

Molecular and Biochemical Detection of Insecticide Resistance in the Leishmania Vector, Phlebotomus papatasi (Diptera: Psychodidae) to Dichlorodiphenyltrichloroethane and Pyrethroids, in Central Iran.

The aim of the present study was to explore resistance markers and possible biochemical resistance mechanisms in the Phlebotomine sand fly Phlebotomus papatasi in Esfahan Province, central Iran. Homogenous resistant strains of sand flies were obtained by exposing P. papatasi collected from Esfahan to a single diagnostic dose of DDT. The adults from the colony were tested with papers impregnated with four pyrethroid insecticides: Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15%, and Lambdacyhalothrin 0.05% to determine levels of cross-resistance. To discover the presence of mutations, a 440 base pair fragment of the voltage gated sodium channel (VGSC) gene was amplified and sequenced in both directions for the susceptible and resistant colonies. We also assayed the amount of four enzymes that play a key role in insecticide detoxification in the resistant colonies. A resistance ratio (RR) of 2.52 folds was achieved during the selection of resistant strains. Sequence analysis revealed no knockdown resistance (kdr) mutations in the VGSC gene. Enzyme activity ratio of the resistant candidate and susceptible colonies were calculated for α-esterases (3.78), β-esterases (3.72), mixed function oxidases (MFO) (3.21), and glutathione-S-transferases (GST) (1.59). No cross-resistance to the four pyrethroids insecticides was observed in the DDT resistant colony. The absence of kdr mutations in the VGSC gene suggests that alterations in esterase and MFO enzymes are responsible for the resistant of P. papatasi to DDT in central Iran. This information could have significant predictive utility in managing insecticide resistant in this Leishmania vector.

Investigation of FMS-Like Tyrosine Kinase 3 Mutation Frequency in Myelodysplastic Syndrome

Introduction: FMS-Like Tyrosine Kinase Class 3 (FLT3) mutations harbor poor prognosis, high relapse, and decreased overall survival in acute myeloblastic leukemia (AML). This mutation is also known to be demonstrated in myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia and acute lymphoblastic leukemia. This study included 94 MDS-diagnosed patients and we tried to investigate FLT3 mutation frequency (as tyrosine kinase domain-TKD and internal tandem duplication-ITD).&#x0D; Materials and Methods: Polymerase chain reaction (PCR), restriction fragment length polymorphism, and agarose-gel electrophoresis methods were used to analyze the mutation. The blood samples were collected in K3-EDTA tubes, and total DNA was isolated using genomic DNA isolation kits (GeneMark, Cat No: DP023P). For the detection of FLT3-ITD mutation, PCR was performed to amplify a 330- base pair fragment of exons 11 and 12 of FLT3 using FAM (Carboxyfluorescein)-labeled ITD-11F and HEX (Hexachloro-Fluorescein )-labeled ITD-12R primers in a thermal cycler (Eppendorf). Similarly, to detect D835 mutation, a 115- bp region of exon 17 of the FLT3 gene region was amplified using primers.&#x0D; Results: One patient was found FLT3-ITD positive (1.1%). The patient was 64-year-old and diagnosed with MDS-excess blast type 2 according to the World Health Organisation 2016 myeloid neoplasm classification. He transformed to AML within 19 months and subsequently died after 1 month. No patient with tyrosine kinase domain mutation was detected.&#x0D; Conclusion: FLT3 mutation is considered a significant parameter to define prognosis in AML. The routine workup of FLT3 screening and the potential of targeting FLT3 inhibition for high-risk MDS may be taken into consideration in the future.

Molecular detection and prevalence of Theileria ovis and Anaplasma marginale in sheep blood samples collected from Layyah district in Punjab, Pakistan.

Theileria ovis and Anaplasma marginale are intracellular pathogens affecting a wide range of animals, causing huge economic losses worldwide. The present study reports the molecular evidence of Theileria ovis and Anaplasma marginale in sheep blood samples (N = 218) collected from Layyah district in Punjab (Pakistan), where economy heavily relies on livestock. A 520 base pair fragment specific for 18S ribosomal RNA gene of Theileria ovis was PCR amplified in 23/218 (10.6%) sheep blood samples, while for Anaplasma marginale, a 265 base pair fragment specific for msp1b gene was generated in 15/218 (6.9%) sheep blood samples. Two blood samples were found co-infected (0.9%) with both parasites. Amplified PCR products of both parasites were confirmed by DNA sequencing and submitted to GenBank. Prevalence of both Theileria ovis (p = 0.3) and Anaplasma marginale (p = 0.4) varied non-significantly among the investigated sheep breeds. Tick burden on dogs present with sheep herds was found associated with Theileria ovis infection in sheep (p = 0.05). It was observed that lambs (p = 0.009), sheep in small herds (p = 0.04), and tick burden on dogs present with sheep herds (p = 0.01) were associated with Anaplasma marginale infection in sheep during the present study. In conclusion, we are reporting a higher prevalence of Theileria ovis than Anaplasma marginale in blood samples of sheep collected from Layyah district. Tick-infested dogs were found to be risk factors for the transmission of both pathogens in sheep, and tick control strategies should be extended to dogs associated with sheep herds in this area.

Seasonal Variability in the Zooplankton Community Structure in a Sub-Arctic Fjord as Revealed by Morphological and Molecular Approaches

Phyto- and zooplankton in Arctic and sub-Arctic seas show very strong seasonal changes in diversity and biomass. Here we document the seasonal variability in the mesozooplankton community structure in a sub-Arctic fjord in Northern Norway based on monthly sampling between November 2018 and February 2020. We combined traditional morphological zooplankton identification with DNA metabarcoding of a 313 base pair fragment of the COI gene. This approach allowed us to provide the most detailed mesozooplankton species list known for this region across an entire year, including both holo- and meroplankton. The zooplankton community was dominated by small copepods throughout the sampling period both in terms of abundance and relative sequence counts. However, meroplankton was the most diverse group, especially within the phylum polychaeta. We identified four distinct periods based on the seasonal analysis of the zooplankton community composition. The pre-spring bloom period (February–March) was characterized by low abundance and biomass of zooplankton. The spring bloom (April) was characterized by the presence of Calanus young stages, cirripedia and krill eggs. The spring-summer period (May–August) was characterized by a succession of meroplankton and a relatively high abundance of copepods of the genus Calanus spp. Finally, the autumn-winter period (September–December) was characterized by a high copepod diversity and a peak in abundance of small copepods (e.g., Oithona similis, Acartia longiremis, Pseudocalanus acuspes, Pseudocalanus elongatus, Pseudocalanus moultoni, Pseudocalanus minutus). During this period, we also observed an influx of boreal warm-water species which were notably absent during the rest of the year. Both the traditional community analysis and metabarcoding were highly complementary and with a few exceptions showed similar trends in the seasonal changes of the zooplankton community structure.

First report regarding molecular epidemiology and novel variant identification of Anaplasma centrale in cattle from Pakistan.

Anaplasma centrale (A. centrale) is an obligate red blood cell residing tick transmitted rickettsiae that has not been studied extensively for its prevalence in cattle along with its epidemiology. Aim of this investigation was to report the seasonal prevalence, phylogeny and epidemiological parameters associated with the prevalence of A. centrale in cattle breeds enrolled from District Layyah in Punjab, Pakistan. A total of 844 blood samples [Cross breed = 300, Holstein Friesian = 244, Sahiwal breed = 300)] were collected from apparently healthy cattle along with epidemiological data during 2017–18. PCR amplified 426 base pair fragment from 16S rRNA gene of A. centrale in 14.4% (122/844) of cattle. Amplified 16S rRNA partial gene sequence of A. centrale were confirmed by DNA sequencing and deposited to GenBank. Highest A. centrale prevalence was observed in spring (24%) followed by autumn (12.4%) summer (10%) and winter (7.1%) seasons. Sahiwal breed (18.3%) was most susceptible to A. centrale infection followed by cross (12.3%) and Holstein Friesian breed (12.3%). 69/844 (8.2%) of Giemsa stained cattle blood smears were also found positive for Anaplasma spp. Farms where animal use to drink pool water and farms where dogs and other dairy animals were living with cattle had higher A. centrale prevalence. Female cattle and dogs having tick burden were found associated with A. centrale infection. Hematological profile was severely disturbed in A. centrale positive cattle. It is recommended that A. centrale should be screened in cattle, in addition to A. marginale, for the effective control of tick born diseases in Pakistan.

High Throughput T Cell Receptor Sequencing Augments Diagnosis and Response Assessment in Patients with Angioimmunoblastic T Cell Lymphoma

Diagnosis and response assessment to therapy has been shown to be challenging in patients with angioimmunoblastic T cell lymphoma (AITL). While at disease onset histology of neoplastic T cell infiltrates in affected lymph nodes and/or bone marrow might be hard to distinguish from reactive lymphproliferation, clonality analyses using T cell receptor (TCR) spectratyping has been helpful in the diagnosis of malignant lymphoma. Nevertheless, resolution of this method is confined to a particular base pair fragment length, which could include several different T cell clones, making detection of minimal residual disease (MRD) almost impossible. From a clinical point of view, rapid responses of AITL to chemotherapy is not uncommon. But as a matter of fact, clinical symptoms of the lymphoma might persist, while neither imaging nor histology is able to detect residual disease.In an attempt to augment diagnosis of malignancy and improve MRD detection in AILT we established a high throughput TCR profiling approach using next generation sequencing (NGS). In order to determine lymphomaspecific TCR gamma V-J rearrangements, we analysed the entire expressed human TCR gamma repertoire in several different tissues using LymphoTrack TRG assays (Invivoscribe) together with the MiSeq Illumina platform.In all analysed cases of histologically proven AITL (n=6), TCR profiling resulted in detection of at least one lymphomaspecific clonal V-Jgamma rearranged TCR sequence in all examined relevant tissue probes (lymph nodes, bone marrow, blood) with observed frequencies up to 16% of all reads in lymph nodes and 7% in blood/bone marrow at the time of diagnosis. Lymphomaspecific TCR sequences as revealed by NGS correlated with clonality results as determined from Vgamma fragments analyses by spectratyping. In addition to confirmation of diagnosis, in some cases lymphomaspecific TCR sequences could be detected in lymph node and bone marrow biopsies up to one year before establishment of AITL diagnosis, in which routine pathology examination could only describe an unspecific lymphadenitis. Treatment responses resulted in diminished frequencies of the lymphomaspecific TCR sequences in blood and bone marrow samples with complete disappearance after allogeneic stem cell transplantation. Of particular importance was the observed sustained detection of lymphomaspecific TCR sequences in bone marrow samples in apparently successful treated patients with refractory clinical symptoms, in which lymphoma activity could not firmly be proven by other imaging or pathology methods.In conclusion, NGS based TCR profiling using V-J gamma rearrangments is very helpful for further analyses of AITL biology and might serve as MRD marker for therapy guidance. DisclosuresNo relevant conflicts of interest to declare.

Genetic Analysis of African Swine Fever Virus From the 2018 Outbreak in South-Eastern Burundi.

African swine fever (ASF) is a contagious viral disease that causes high mortality, approaching 100%, in domestic pigs and wild boars. The disease has neither a cure nor a vaccine, and it is caused by an ASF virus (ASFV), the only member of the family Asfarviridae, genus Asfivirus, and the only known DNA arbovirus. Twenty-four genotypes of ASFV have been described to date, and all of them have been described in Africa. ASF is endemic in Burundi, and several outbreaks have been reported in the country; the disease continues to economically impact on small-scale farmers. This study aimed at genetic characterization of ASFV that caused an ASF outbreak in the Rutana region, Burundi, in the year 2018. Tissue samples from domestic pigs that died as a result of a severe hemorrhagic disease were collected in order to confirm the disease using polymerase chain reaction (PCR) and to conduct partial genome sequencing. Nucleotide sequences were obtained for the B646L (p72) gene, the intergenic fragment between the I73R and I329L genes, and the central variable region (CVR) of the B602L gene. Phylogenetic analysis of the Burundian 2018 ASFV grouped the virus within B646L (p72) genotype X and clustered together with those reported during the 1984 and 1990 outbreaks in Burundi with high nucleotide identity to some ASFV strains previously reported in neighboring East African countries, indicating a regional distribution of this ASFV genotype. Analysis of the intergenic fragment between I73R and I329L genes showed that the Burundian 2018 ASFV described in this study lacked a 32–base pair (bp) fragment present in the reference genotype X strain, Kenya 1950. In addition, the strain described in this study had the signature AAABNAABA at the CVR (B602L) gene and showed 100% amino acid sequence identity to viruses responsible for recent ASF outbreaks in the region. The virus described in this study showed high genetic similarities with ASFV strains previously described in domestic pigs, wild suids, and soft ticks in East African countries, indicating a possible common wild source and continuous circulation in domestic pigs in the region.