Sequencing Of PCR Products Research Articles

Genetic variants of CSN1S1, CSN2, CSN3, and BLG genes and their association with dairy production traits in Sahiwal cattle and Nili-Ravi buffaloes

Milk protein genes are associated with milk yield and composition in dairy animals. The present study aimed to identify milk protein genes (CSN1S1, CSN2, CSN3, and BLG) genetic variants and their association with milk yield in Sahiwal cattle and Nili-Ravi buffaloes. One hundred animals from each species were selected to collect blood samples and milk production records. Primers were designed for these milk protein genes for PCR amplification. Sequencing of resultant PCR products revealed a higher number of SNPs (13 vs. 7, 5 vs. 1, and 6 vs. 2) in Sahiwal as compared to Nili-Ravi animals in CSN1S1, CSN2, and CSN3 genes, respectively. However, a single SNP was observed in BLG gene of both species. Association analysis revealed that one SNP in BLG gene of Nili-Ravi was associated (p < 0.05) with 305-day milk yield. Two SNPs at CSN1S1 gene in Sahiwal were associated with dry-period. Similarly, one SNP at CSN1S1 and two SNPs at CSN3 gene showed significant association (p < 0.05) with average calving-interval in Sahiwal while two SNPs in CSN1S1 gene were associated (p < 0.05) with this trait in Nili-Ravi. These SNPs could be helpful as candidate variants for marker-assisted selection in cattle and buffaloes for improvement of lactation performance.

Using next generation sequencing for molecular detection and differentiation of Anaplasma phagocytophilum variants from host seeking Ixodes scapularis ticks in the United States

Anaplasmosis is increasingly common in the United States, with cases being reported over an expanding geographic area. To monitor for changes in risk of human infection, the U.S. Centers for Disease Control and Prevention monitors the distribution and abundance of host-seeking vector ticks (Ixodes scapularis and Ixodes pacificus) and their infection with Anaplasma phagocytophilum. While several variants of A. phagocytophilum circulate in I. scapularis, only the human-active variant (Ap-ha) appears to be pathogenic in humans. Failure to differentiate between human and non-human variants may artificially inflate estimates of the risk of human infection. Efforts to differentiate the Ap-ha variant from the deer variant (Ap-V1) in ticks typically rely on traditional PCR assays coupled with sequencing of PCR products. However, laboratories are increasingly turning to Next Generation Sequencing (NGS) to increase testing efficiency, retain high sensitivity, and increase specificity compared with traditional PCR assays. We describe a new NGS assay with novel targets that accurately segregate the Ap-ha variant from other non-human variants and further identify unique clades within the human and non-human variants. Recognizing that not all investigators have access to NGS technology, we also developed a PCR assay based on one of the novel targets so that variants can be visualized using agarose gel electrophoresis without the need for subsequent sequencing. Such an assay may be used to improve estimates of human risk of developing anaplasmosis in North America.

Utility of the trnH–psbA region for DNA barcoding of Aconitum anthora L. and related taxa

Aim. The genus Aconitum is represented in the Carpathian region by a significant number of endemic species. Some of these species, in particular A. pseudanthora and A. jacquinii, are included in the Red List of Ukraine. However, their taxonomic status and consequently the importance of conserving their natural resources remain uncertain. DNA barcoding techniques, e.g., the application of molecular markers based on variable regions of the chloroplast genome can help clarify these questions. Methods. DNA isolation, PCR amplification, sequencing of PCR products, phylogenetic analysis. Results. The psbA-trnH intergenic spacer region of A. pseudanthora and A. jacquinii specimens from Western Ukraine was amplified and sequenced. It is shown that the spacer sequences for these two species are identical, but differ in short indels and nucleotide substitutions from the psbA-trnH sequences of A. anthora s.l. specimens from Western Europe and China. Phylogenetic analyzes showed the phylogenetic affinity of the members of the A. anthora complex to the subgenus Lycoctonum. Conclusions. The use of the psbA-trnH intergenic spacer region for DNA barcoding makes it possible to unambiguously distinguish samples of A. jacquinii and A. pseudanthora from western Ukraine from samples of A. anthora s.l. of other geographical origins. The results obtained confirm the reticular nature of the evolution in the genus Aconitum.

Campylobacter jejuni subdural hygroma infection in a 2-year old boy: case report and a brief literature review

BackgroundCampylobacter jejuni is a common cause of acute gastroenteritis, but central nervous system infections are rare manifestations of Campylobacter infection. Therefore, C. jejuni trauma-related subdural hygroma infection in children is poorly described in the literature.Case presentationWe described a 2-year old boy with lobar holoprosencephaly presenting with subdural hygroma following head trauma. C. jejuni infection was confirmed from a subdural hygroma sample by culture as well as by DNA sequencing of a broad range 16S rDNA PCR product. Cerebrospinal fluid from the ventriculoperitoneal shunt remained sterile. Combined neurosurgical and antimicrobial treatment led to complete recovery. Review of the literature showed that the most common manifestation of Campylobacter central nervous system infection is meningitis, mostly in neonates, and subdural hygroma infection was described for only one case.ConclusionsSubdural hygroma infection caused by C. jejuni is a rare clinical condition in children. Molecular methods represent an important tool for the detection of rare or unexpected pathogens. No standard recommendations for antimicrobial treatment of C. jejuni subdural space infection in children are available, but meropenem treatment combined with surgery seems to be an effective approach.

Quantitative reverse transcription PCR assay to detect a genetic marker of pyrethroid resistance in Culex mosquitoes.

Pyrethroid insecticides are widely used to control mosquitoes that transmit pathogens such as West Nile virus (WNV) to people. Single nucleotide polymorphisms (SNP) in the knockdown resistance locus (kdr) of the voltage gated sodium channel (Vgsc) gene in Culex mosquitoes are associated with knockdown resistance to pyrethroids. RNAseq was used to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. The cDNA sequences were used to develop a quantitative reverse transcriptase PCR assay that detects the L1014F kdr mutation in the Vgsc. Because this locus is conserved, the assay was used successfully in six Culex spp. The resulting Culex RTkdr assay was validated using quantitative PCR and sequencing of PCR products. The accuracy of the Culex RTkdr assay was 99%. The L1014F kdr mutation associated with pyrethroid resistance was more common among Cx. pipiens than other Culex spp. and was more prevalent in mosquitoes collected near farmland. The Culex RTkdr assay takes advantage of the RNA that vector control agencies routinely isolate to assess arbovirus prevalence in mosquitoes. We anticipate that public health and vector control agencies may employ the Culex RTkdr assay to define the geographic distribution of the L1014F kdr mutation in Culex species and improve the monitoring of insecticide resistance that will ultimately contribute to effective control of Culex mosquitoes.

Characteristics of Klebsiella Pneumonia St4 Coharboring Qnrb1, Aac-Ib-Cr, CTX-M-15 and SHV 11 in A Tunisian Hospital

Background: Multiresistant Klebsiella pneumonia are predominant cause of hospital acquired infection. This work describes the molecular epidemiology of these isolates in Tunisian Hospital. Methods: Between October 2010 and June 2013, 50 non-duplicated clinical K.pneumoniae were selected based on nalidixic acid (NA) resistance and were characterized. Isolates were identified using APi 20E system. Susceptibility testing was determined using the disc diffusion method and the micro dilution technique to determine the MIC of ciprofloxacin. PMQR and ESBL genes were detected by PCR and positive results were confirmed by direct sequencing of PCR products. Multilocus sequence typing (MLST) was performed to determine the genetic relationship among isolates. Conjugation and transformation were done to know if PMQR and ESBL were carried with one or two plasmids. Results: 20 PMQR harboring K.pneumoniae representing 40% of all NA resistant isolates were characterized. Among PMQR positive K.pneumoniae 13 were resistant to amoxicillin, amoxicillin/clavulanic acid, ticarcilline, piperacillin, cefaloridine, cefotaxime (CTX) and ceftazidime. The rate of resistance to gentamicin, tobramycin and amikacin were 85%, 95% and 25% respectively. Out of 20 K.pneumoniae (60%) were qnr positive (1 qnrA6 and 11 qnrB1) and (60%) were aac-Ib-cr positive. 33.3% harbored the aac-Ib-cr and qnrB1 determinants. Out of all PMQR positive strains, 65% harbored ctx-M-15 gene. It was associated to shv11 in three cases and tem1 in two cases. The predominant types were ST4 (35%) and ST15 (20%). ST 101(15%) and ST 147 (10%) come in second order. one case of each ST14,ST86,ST336 and ST307 were also observed. qnrB1, aac-Ib-cr, Ctx-m-15 can be carried with more than one plasmid in the same bacteria. Conclusion: The co-existing of different genes conferring resistance among the same and different family of antibiotics is a big threat to patient because it limits the therapeutic process. This phenomenon is a problem of concern that needs to improve the resistance surveillance of multi gene carrying K pneumonia.

Identification and Characterization of PTE-2, a Stowaway-like MITE Activated in Transgenic Chinese Cabbage Lines.

Transposable elements (TEs) are DNA fragments that can be replicated or transposed within a genome. TEs make up a high proportion of the plant genome and contribute to genetic diversity and evolution, affecting genome structure or gene activity. Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II DNA transposable elements. MITEs have specific sequences, target site duplications (TSDs), and terminal inverted repeats(TIRs), which are characteristics of the classification of MITE families. In this study, a Stowaway-like MITE, PTE-2, was activated in transgenic Chinese cabbage lines. PTE-2 was revealed by in silico analysis as the putative activated element in transgenic Chinese cabbage lines. To verify the in silico analysis data, MITE insertion polymorphism (MIP) PCR was conducted and PTE-2 was confirmed to be activated in transgenic Chinese cabbage lines. The activation tendency of the copy elements of PTE-2 at different loci was also analyzed and only one more element was activated in the transgenic Chinese cabbage lines. Analyzing the sequence of MIP PCR products, the TSD sequence and TIR motif of PTE-2 were identified and matched to the characteristics of the Stowaway-like MITE family. In addition, the flanking region of PTE-2 was modified when PTE-2 was activated.

First report of Chilli leaf curl India virus (ChiLCINV) infecting Stachytarpheta jamaicensis in India

Blue snakeweed (Stachytarpheta jamaicensis) exhibiting symptoms of virus infection such as crinkling, yellowing, mottling, reduction in leaf size and downward curling were observed during disease survey in Pune, Maharashtra, India in 2021. The presence of a begomovirus in the infected plants was confirmed by PCR assay using universal primers and sequence analysis. BLASTn analysis of the sequence of PCR product revealed that it shared 95.55% identity with Chilli leaf curl India virus (ChiLCINV) isolates. In the phylogenetic analysis, the sequence clustered with ChiLCINV isolates reported from various hosts. To our knowledge, this is the first report of a begomovirus (ChiLCINV) infecting S. jamaicensis in India.

Analysis of sequence polymorphism of OsHKT1;5 gene in rice (Oryza sativa L.)

In salt stress conditions, the excessive accumulation of Na+ ion in the cytoplasm could cause ion toxicity, metabolic imbalances, and reduction in growth of plant. The ability of maintaining a low Na+ concentrations or low Na+/K+ ratio in cytosol plays the important roles of plant adapt to salinity stress. In rice, the HKT transporter family has been proven to be critical for salt tolerance and the maintenance of Na+, K+ homeostasis under salinity conditions. In this study, we conducted the sequence polymorphisms analysis in the OsHKT1;5 coding region in order to detect the potential allelic variants in some local rice cultivars in Viet Nam. The exon 1 region and exon 2-3 region of OsHKT1;5 gene were amplified by PCR in two separate reactions. After direct sequencing of PCR products, the full length coding region of OsHKT1;5 gene was obtained and compared to reference Nipponbare OsHKT1;5 coding sequence. Accordingly, twelve nucleotide substitutions in coding sequence of OsHKT1;5 were identified, including six non-synonymous substitutions (G382A, C418G, G551A, C994G, C1183G, A1630C) and the other six synonymous substitutions (G225A, C1038G, G1152A, G1261A, G1304C, C1608T). Further analysis revealed that six non-synonymous substitutions (G382A, C418G, G551A, C994G, C1183G, A1630C) caused the changes in amino acids (D128N, P140A, R184H, H332D, V395L, and S544R). The single nucleotide polymorphism A1630C leading substitution of S544R was newly found in the Chiem Rong cultivar. In silico analysis of protein structure and post-translational modifications indicated that amino acid substitutions had no influence on protein structure but led to changes in post-translational modifications such as phosphorylation and SUMOylation.

Molecular detection of novel Anaplasma sp . and zoonotic hemopathogens in livestock and their hematophagous biting keds (genus Hippobosca) from Laisamis, northern Kenya.

Background: Livestock are key sources of livelihood among pastoral communities. Livestock productivity is chiefly constrained by pests and diseases. Due to inadequate disease surveillance in northern Kenya, little is known about pathogens circulating within livestock and the role of livestock-associated biting keds (genus Hippobosca) in disease transmission. We aimed to identify the prevalence of selected hemopathogens in livestock and their associated blood-feeding keds. Methods: We randomly collected 389 blood samples from goats (245), sheep (108), and donkeys (36), as well as 235 keds from both goats and sheep (116), donkeys (11), and dogs (108) in Laisamis, Marsabit County, northern Kenya. We screened all samples for selected hemopathogens by high-resolution melting (HRM) analysis and sequencing of PCR products amplified using primers specific to the genera: Anaplasma, Trypanosoma, Clostridium, Ehrlichia, Brucella, Theileria, and Babesia. Results: In goats, we detected Anaplasma ovis (84.5%), a novel Anaplasma sp. (11.8%), Trypanosoma vivax (7.3%), Ehrlichia canis (66.1%), and Theileria ovis (0.8%). We also detected A. ovis (93.5%), E. canis (22.2%), and T. ovis (38.9%) in sheep. In donkeys, we detected ' Candidatus Anaplasma camelii' (11.1%), T. vivax (22.2%), E. canis (25%), and Theileria equi (13.9%). In addition, keds carried the following pathogens; goat/sheep keds - T. vivax (29.3%) , Trypanosoma evansi (0.86%), Trypanosoma godfreyi (0.86%), and E. canis (51.7%); donkey keds - T. vivax (18.2%) and E. canis (63.6%); and dog keds - T. vivax (15.7%), T. evansi (0.9%), Trypanosoma simiae (0.9%) , E. canis (76%), Clostridium perfringens (46.3%), Bartonella schoenbuchensis (76%), and Brucella abortus (5.6%). Conclusions: We found that livestock and their associated ectoparasitic biting keds carry a number of infectious hemopathogens, including the zoonotic B. abortus. Dog keds harbored the most pathogens, suggesting dogs, which closely interact with livestock and humans, as key reservoirs of diseases in Laisamis. These findings can guide policy makers in disease control.

Identification and phylogenetic characterization based on DNA sequences from RNA ribosomal genes of thermophilic microorganisms in a high elevation Andean tropical geothermal spring

Several microorganisms can survive in harsh acid environments in geothermal springs at high temperatures across the Equatorial Andes Mountains. However, little is known about their physiological features and phylogenetic composition. Here we identify thermophilic microorganisms (bacteria, fungi, and microalgae) hosted in an almost unexplored geothermal spring (known as “Aguas Hediondas”). The phylogeny of the cultures was determined by analyzing physiological features and DNA sequences of PCR products for 16S rRNA, ITS, and 23S rRNA genes. Twenty pure cultures were isolated from the samples, including 17 for bacteria, one for cyanobacterium, one for eukaryotic microalgae, and one for fungus. Most bacterial strains were gram-positive, spore-forming, and bacilli (Bacillus). Cyanobacterium strain belonged to Chroococcidiopsis and the eukaryotic microalgae to Chlorophyta. The unique fungal strain isolated was closely related to T. duponti. Through our study, isolated thermophilic bacteria, microalgae and fungi from the “Aguas Hediondas” geothermal spring were characterized and identified. This study represents one of the first extensive molecular characterizations of extremophile microbes in the Tropical Equatorial Andes. Keywords. microbial diversity; DNA markers; extremophiles; phylogenetics

PM 7/20 (3) Erwinia amylovora

PM 7/20 (3) <i>Erwinia amylovora</i>

Development and evaluation of a loop-mediated isothermal amplification (LAMP) technique for rapid, accurate, and specific detection of Blastocystis spp. in AIDS patients.

Blastocystis spp. is one of the most prevalent intestinal parasites with worldwide distribution. Various diagnostic methods with different sensitivities and specificities have been used to detect Blastocystis in clinical samples. The present study aims to develop and evaluate a LAMP assay to detect Blastocystis spp. in AIDS patients for the first time. In this cross-sectional study, 98 AIDS patients with an average CD4 + T lymphocyte count lower than 150 cells/mm3 participated in the study. The presence of Blastocystis spp. in the stool samples of AIDS patients was examined by parasitology (direct wet mount and concentration assays) and molecular (PCR and LAMP) methods. The 18 SSU rRNA genomic target was used to design the specific primers for the PCR and LAMP assays. The specificity of designed primers for the LAMP assay was evaluated using the sequencing of a conventional PCR product by the external LAMP primers. The data were analyzed using the SPSS software and chi-square test and Fischer's exact tests were used and Cohen's Kappa calculates the agreement of the molecular tests. Associations were tested using odd ratios (OR) and 95% confidence intervals (CI) after adjustments. Out of 98 stool samples from patients with AIDS, 9 (9.18%), 13 (13.26%), and 15 (15.30%) samples were detected positive for Blastocystis spp. by parasitology, PCR, and LAMP techniques, respectively. PCR amplification and subsequent sequencing of the product sequences revealed that the obtained partial sequences were identical to the corresponding 18 SSU rRNA sequences reported in GenBank. The higher positivity rate for Blastocystis spp. among studied AIDS patients by LAMP technique compared to other diagnostic methods showed the higher potential and effectiveness of this relatively new described molecular assay for the detection of Blastocystis spp. in AIDS patients. The accurate and rapid detection of emerging intestinal protozoa such as Blastocystis is of clinical importance for better prevention and timely treatment of the disease, especially in immunocompromised patients. The results obtained for the first time showed that the sensitivity and accuracy of the LAMP technique in the diagnosis of Blastocystis spp. in AIDS patients is very high.

Molecular marker-based characterization of cytoplasm and restorer of male sterility (Ms) locus in commercially grown onions in India.

The cytoplasmic-genic male-sterility system has been extensively employed for the production of onion hybrids. Molecular marker-assisted characterization of the cytotypes and genotyping at the restorer-of male-fertility (Ms) locus is important for the accelerated breeding of onion hybrids. Indian onion breeding has focussed more on open-pollinated varieties than hybrids. To accelerate the breeding efforts, marker-assisted selection (MAS) plays a pivotal role. This study aimed to characterize the Indian breeding lines, varieties, hybrids, and exotic accessions for cytotype and Ms locus. For cytoplasm, cytotype markers, accD, and MKFR and for Ms locus identification, PCR markers AcPMS1 and AcSKP1 were employed. Bulk strategy to identify cytoplasm and Ms locus was tested. Sequencing of PCR products amplified by accD was also tried. Both the accD and MKFR were synonymous in cytoplasm identification except in T821 where T cytoplasm was identified. AcPMS1 was more reliable than AcSKP1 for Ms locus identification. Sequencing proved that N and T cytoplasm are identical. Bulking strategy can be used for cytotype identification but not for Ms locus. Indian onions have a predominance of normal (N) cytoplasm and homozygous recessive (msms) locus. This might be beneficial for hybrid development. S cytoplasm was identified in exotic varieties. For the first time, T cytoplasm has been reported from India. These findings will assist Indian onion breeders to develop MAS strategies for accelerating hybrid development programs. And for the release of onion hybrids with high productivity and uniformity.

Targeting Mutational Landscape of TP53 in patients diagnosed with Oral Cancer living in Senegal

Introduction Genomic mutations in TP53 gene in association with etiological risk factors have been associated with oral carcinogenesis. Herein, we screened for genomic variants of TP53 predisposing to oral cancers in Senegalese patients. Methodology 88 patients with confirmed diagnostic were recruited after informed consent. Blood samples were collected from each patient to perform DNA extraction, PCR amplification of all coding exons of TP53 followed by Sanger Sequencing of PCR products. Nucleotide sequences were analysed with Genalys software. 94 blood donors with no cancer diagnosis were also recruited as controls for association study between the most common variants identified in patients and predisposition to oral cancers. Results Sequence analysis showed that 52.27% of patients carry at least one mutation in TP53. Eleven genomic variants were identified, 7 variants already reported in databases and 4 new variants. The most recurrent variants in this study already reported as cancer-related variants were Pro72Arg (rs1042522; Arginine frequency estimated at 31.26%) and a 16 bp insertion in intron 3 (rs59758982; allelic frequency estimated at 26.25%). Haplotype analysis between these variants showed a strong linkage disequilibrium (D’ = 0.999, r2 = 0.153 and p-value &lt; 0.05). However, association study did not find any significant association with susceptibility to oral cancer (p-value &gt; 0.05). Conclusion Our study highlighted that despite the absence of association between the two most common cancer-related variants in Senegalese patients diagnosed with oral cancer, their strong LD suggested that they could be transmitted together in a common haplotype which may be implicated in oral carcinogenesis.

Complete genome sequence of vallota mosaic virus detected in a narcissus bulb imported from the United States to Japan.

Narcissus (Narcissus albidus) imported from the United States exhibited leaf chlorosis during post-entry quarantine. We employed next-generation sequencing (NGS) on symptomatic leaf samples and detected vallota mosaic virus (ValMV), belonging to the genus Potyvirus, family Potyviridae, as the viral agent. Sanger sequencing of PCR products and rapid amplification of cDNA ends based on NGS contigs revealed that ValMV is 9,451 nucleotides (nt) in length, excluding the poly(A) tail. Nucleotide and amino acid (aa) sequences of the coat protein region had over 98% identity to previously reported ValMV isolates. In each of the 10 regions encoding mature proteins, however, the sequence identity to other potyviruses was 49.5-71.9% nt and 18.3-78.9% aa, values that are below the species demarcation thresholds for the family Potyviridae. Phylogenetic analysis revealed that our ValMV isolate is most closely related to known ValMV isolates and is grouped with other potyviruses. Taken together, our results indicate that the newly isolated ValMV belongs to a distinct species in the genus Potyvirus. This study provides the first report of the complete ValMV genome sequence and the first record of this virus in narcissus.

C1: Targeting mutational landscape of TP53 gene in patients diagnosed with oral cancers living in Senegal

INTRODUCTION: Looking for the mutational landscape on a genomic scale has provided in the last few years unprecedented perspectives for understanding molecular pathogenesis of cancer progression, response to treatment and genetic predisposition. In oral cancers, numerous genomic studies have been devoted to alterations in the TP53 gene and their clinical significance in carcinogenesis. Herein, we screened for genomic variants of TP53 predisposing to oral cancers in Senegalese patients. MATERIALS AND METHODS: Eighty-eight patients with confirmed diagnostic were recruited after informed consent from Stomatology Unit of Aristide le Dantec Hospital. Blood samples were collected from each patient to perform DNA extraction, PCR amplification of all coding exons of TP53 followed by Sanger Sequencing of PCR products. Nucleotidic sequences were analyzed with Genalys software. RESULTS: Mean age at diagnosis was estimated at 51.9 ± 17.98 years with a sex-ratio of 0.76. The majority of patients (64/88) had a negative alcohol-tobacco status. Tumor histology showed that 90% of patients had squamous cell carcinoma localised preferentially on the tongue (22.7%), gum (20.5%) and cheek mucosa (19.3%). Mutation screening of TP53 showed that 52.27% of the patients carried at least one mutation. Eleven genomic variants were identified, seven already reported in mutation databases and four new variants. The most recurrent variants was p.Pro72Arg (rs1042522; Arginine allelic frequency estimated at 31.26%) and a 16 bp deletion in intron 3 (rs59758982; allelic frequency estimated at 26.25%). Linkage disequilibrium (LD) calculation has shown that these two variants are in strong LD but tests of association with controls show that they could not predispose towards oral cancers. CONCLUSION: The results of the present investigation suggest that the two most common variants found in Senegalese patients are not predisposing to oral cancers. Further comprehensive investigations on reported and new variants found in this study should be conducted to highlight their functional impacts and hypothetic oncogenic associated function.

Clinically Manifesting, Naturally Occurring Fowl Glioma in a Leghorn Chicken in Germany.

Fowl glioma-inducing virus (FGV), a strain of avian leukosis virus (ALV) subgroup A, is the causal agent of fowl glioma characterized by multiple nodular astrocytic growths, gliosis, and lymphocytic encephalitis. Also associated with FGV infection are cases of cerebellar hypoplasia, perineuromas, and nonsuppurative myocarditis. Though fowl glioma has been recognized in several countries, most reports of FGV infection come from Japan. A 9-mo-old brown leghorn from a German farm with nine leghorns was presented to a veterinarian with an impaired general health with torticollis, tremor, and incoordination. Histopathology revealed multifocal nodular astrocytic growths, gliosis, and a lymphoplasmacytic encephalitis. Immunohistochemically, neoplastic astrocytes showed positivity for anti-ALV antibody. FGV was detected in the brain with nested reverse transcription-polymerase chain reaction (RT-PCR) and subsequent sequencing of PCR product. The remaining eight birds were screened for the presence of ALV with real-time RT-PCR. Four leghorns tested positive for exogenous ALV in nested RT-PCR with an identical nucleotide sequence as the leghorn with neurological symptoms. To the authors' knowledge this is the first report of a natural FGV infection in a brown leghorn in Germany with clinical manifestation.

First insights on the diversity of the genus Ostreopsis (Dinophyceae, Gonyaulacales) in Guadeloupe Island, with emphasis on the phylogenetic position of O. heptagona

The present study aims to identify epiphytic Ostreopsis cells collected in Guadeloupe between 2017 and 2018 using a morpho-molecular approach. This method combined microscopical observations of wild specimens (light and scanning electron microscopy) with a phylogenetic analysis inferred from concatenated sequences of ribosomal operon (SSU + ITS + LSU) of Ostreopsidoideae. Four distinct morphotypes were identified in our samples and studied by SEM. Molecular data obtained from single-cell PCR for the four morphotypes were consistent with observations and confirmed the presence of three Ostreopsis species resolved in well characterized genotypes (O. cf. ovata, O. lenticularis and O. siamensis) and an unidentified clade. Detailed morphological characters including sulcal plates confirmed the identification of the last morphotype as O. heptagona D.R. Norris, J.W. Bomber & Balech, which forms a new basal clade in the genus, not previously reported. Observations highlighted overlapping sizes for O. lenticularis, O. siamensis and O. heptagona. Direct sequencing of PCR products obtained for some cells of O. lenticularis and O. heptagona collected at one site revealed unexpectedly the presence of the parasitoid dinoflagellate Amoebophrya. Some Ostreopsis cells were found partially emptied and exhibiting a compact mass. Further analyses are needed to understand the ecological role of Amoebophrya on blooms of epiphytic Ostreopsis species.

Evaluation of the Role of the KRAS Gene Polymorphism LCS6 (rs61764370) in Iraqi Women with Ovarian Cancer.

In carcinogenesis, KRAS is an essential oncogene that plays a key function. The polymorphism of rs61764370 is a candidate for cancer susceptibility in KRAS3' untranslated region. The current study aimed to determine the role and impact of the KRAS gene polymorphism (rs61764370 T>G) on the risk of ovarian cancer development in the Iraqi population. In total, 84 ovarian cancer patients and 28 ovarian benign tumors were involved in a case-control study. DNA extraction from the formalin-fixed/paraffin-embedded tissues, followed by the sequencing of PCR products was carried out in genetic analysis for the detection of the KRAS polymorphism (rs61764370 T>G). The results showed that the frequencies of the KRAS gene polymorphism (rs61764370) in ovarian cancer patients were 78 (92.85%) and 6 (7.15%) with genotypes homozygote TT and heterozygote TG, respectively. These corresponding values in patients with benign ovarian tumors were 25 (89.3%) and 3 (10.7%) with homozygote TT and heterozygote TG, respectively. None of the patients either with malignant or benign tumors have been detected with homozygote genotype GG. Genotype frequency of the TT and TG showed that the heterozygote TT genotype vs. TG and T allele vs. G allele were more frequent in malignant and benign tumors (P≤0.01). Statistically, there was no association between the KRAS polymorphism and the clinical characteristics of ovarian cancer patients, such as age, family history, menopause, histological type, tumor size, or tumor stage. In conclusion, a significant association was found between rs61764370 and the risk of ovarian cancer in the Iraqi population, particularly those with genotypes homozygote TT. On the other hand, genotypes had no relationship with any of the clinical characteristics of ovarian cancer patients. Additional well-designed studies with larger sample sizes are recommended to validate the precise role of KRAS LCS6 variations in ovarian cancer risk.