The study aimed to investigate molecularly the presence of flea-borne viruses in infested small ruminants with fleas. It was carried out in Egypt\'s Northern West Coast (NWC) and South Sinai Governorate (SSG). Three specific primers were used targeting genes, ORF103 (for Capripoxvirus and Lumpy skin disease virus), NS3 (for Bluetongue virus), and Rdrp (for Coronavirus), followed by gene sequencing and phylogenetic analyses. The results revealed that 78.94% of sheep and 65.63% of goats were infested in the NWC area, whereas 49.76% of sheep and 77.8% of goats were infested in the SSG region. Sheep were preferable hosts for flea infestations (58.9%) to goats (41.1%) in the two studied areas. Sex and age of the animals had no effects on the infestation rate (p > 0.05). The season and site of infestation on animals were significantly different between the two areas (p < 0.05). Ctenocephalides felis predominated in NWC and Ctenocephalides canis in SSG, and males of both flea species were more prevalent than females. Molecular analysis of flea DNA revealed the presence of Capripoxvirus in all tested samples, while other viral infections were absent. Gene sequencing identified three isolates as sheeppox viruses, and one as goatpox virus. The findings suggest that Capripoxvirus is adapted to fleas and may be transmitted to animals through infestation. This underscores the need for ongoing surveillance of other pathogens in different regions of Egypt.

Previously, we demonstrated unique insertion/deletion polymorphisms of equine histidine-rich glycoprotein (eHRG) with five genotypes composed of 45-bp or 90-bp deletions in the histidine-rich region of eHRG in Thoroughbred horses. Although leukocytes are typically used to collect DNA for genotyping, blood sampling from animals is sometimes difficult and invasive. Moreover, the method for extracting DNA from blood leukocytes involves complicated steps and must be performed soon after blood sampling for sensitive gene analysis. In the present study, we performed eHRG genotyping using DNA, isolated from oral mucosa swabs collected by rubbing the mucosa on the underside of the upper lip of horses and 100 mg of freshly excreted feces obtained by scraping their surface. In the present study, we performed eHRG genotyping using DNA isolated from oral mucosa swabs and feces of horses (18 Thoroughbreds, 17 mixed breeds, 2 warm bloods), and compared the accuracy of this method with that of the method using DNA from leukocytes. The DNA derived from oral mucosa swabs was sufficient in quantity and quality for eHRG genotyping. However, DNA derived from fecal samples requires a more sensitive detection system because of contamination with non-horse DNA, and the test quality is low. Collection of oral mucosa swabs is less invasive than blood sampling; further, oral swabs can be stored for a longer period in a specified high-quality solution. Therefore, collecting DNA samples from oral mucosa swabs is recommended for the genetic analysis of not only horses but also other animals that are not accustomed to humans.

Single-nucleotide polymorphisms (SNPs) can serve as reliable markers in genetic engineering, selection, screening examinations, and other fields of science, medicine, and manufacturing. Whole-genome sequencing and genotyping by sequencing can detect SNPs with high specificity and identify novel variants. Nonetheless, in situations where the interest of researchers is individual specific loci, these methods become redundant, and their cost, the proportion of false positive and false negative results, and labor costs for sample preparation and analysis do not justify their use. Accordingly, accurate and rapid methods for genotyping individual alleles are still in demand, especially for verification of candidate polymorphisms in analyses of association with a given phenotype. One of these techniques is genotyping using TaqMan allele-specific probes (TaqMan dual labeled probes). The method consists of real-time PCR with a pair of primers and two oligonucleotide probes that are complementary to a sequence near a given locus in such a way that one probe is complementary to the wild-type allele, and the other to a mutant one. Advantages of this approach are its specificity, sensitivity, low cost, and quick results. It makes it possible to distinguish alleles in a genome with high accuracy without additional manipulations with DNA samples or PCR products; hence the popularity of this method in genetic association studies in molecular genetics and medicine. Due to advancements in technologies for the synthesis of oligonucleotides and improvements in techniques for designing primers and probes, we can expect expansion of the possibilities of this approach in terms of the diagnosis of hereditary diseases. In this article, we discuss in detail basic principles of the method, the processes that influence the result of genotyping, criteria for selecting optimal primers and probes, and the use of locked nucleic acid modifications in oligonucleotides as well as provide a protocol for the selection of primers and probes and for PCR by means of rs11121704 as an example. We hope that the presented protocol will allow research groups to independently design their own effective assays for testing for polymorphisms of interest.
Однонуклеотидные полиморфизмы (SNP) могут служить надежными маркерами в генной инженерии, селекции, скрининговых обследованиях и других областях науки, медицины и производства. Полногеномное секвенирование и генотипирование при помощи секвенирования могут высокоспецифично детектировать SNP и выявлять новые аллели. Однако в ситуациях, когда интерес исследователей направлен на отдельные конкретные локусы, эти методы становятся избыточными, а их цена, доля ложноположительных и ложноотрицательных результатов и трудозатраты на пробоподготовку и анализ не оправдывают их применения. Поэтому точные и быстрые методы генотипирования отдельных аллелей все еще остаются востребованными, особенно при проверке кандидатных полиморфизмов в анализах ассоциации с определенным фенотипом. Один из таких методов – генотипирование с использованием аллель-специфичных зондов TaqMan (TaqMan dual labeled probes). Метод заключается в реакции ПЦР в реальном времени с использованием пары праймеров и двух олигонуклеотидных зондов, комплементарных последовательности вблизи данного локуса таким образом, что один зонд комплементарен аллелю дикого типа, а другой – мутантному аллелю. Преимущества метода заключаются в его специфичности, чувствительности, невысокой стоимости и быстроте получения результатов. Он позволяет с высокой точностью различать аллели в геноме в одностадийной ПЦР без дополнительного этапа разделения продуктов реакции, что делает его востребованным в исследованиях генетических ассоциаций в молекулярной генетике и медицине. Благодаря развитию технологий синтеза олигонуклеотидов и совершенствованию методов подбора праймеров и зондов можно ожидать расширения возможностей применения этого подхода в диагностике наследственных заболеваний. В настоящей статье мы разобрали основные принципы метода, процессы, влияющие на результат генотипирования, критерии подбора оптимальных праймеров и зондов, использование LNA-модификаций в олигонуклеотидах, а также привели протокол подбора праймеров, зондов и ПЦР на примере SNP rs11121704. Мы надеемся, что представленный протокол позволит исследовательским группам самостоятельно подбирать собственные эффективные тест-системы для проверки интересующих полиморфизмов.

Parvovirus infection affects several animal species, especially young animals. In birds, parvovirus infection has been described in Muscovy ducks, turkeys, and chickens, all of which had enteric diseases characterized by diarrhea. Chicken parvovirus (ChPV) has been detected in poultry around the world in animals affected by enteric problems, showing dwarfism, cloacal pasting, and diarrhea. In Brazil, ChPV was detected in chickens affected by diarrhea fifteen years ago. However, the genetic characteristics of ChPV circulating in chicken flocks were not determined. Therefore, the aim of the present investigation was to determine the genetic characteristics of the VP1 gene from ChPV detected in chickens affected by enteric diseases in Brazil. For this purpose, a molecular approach was used. Specific primers were designed to flank the complete VP1 gene of ChPV and amplify it using PCR. The amplified products from samples of chickens with enteric diseases were sequenced, and 22 complete CDs of the VP1 gene were obtained. These samples, compared to the ABU-P1 sequence, showed 17 sequences with high nucleotide (NT) similarity of 92.7-97.4% and amino acid (AA) similarity of 94.8-99.5% associated with Runting and Stunting syndrome (RSS); there were also five samples associated with hens with diarrhea with unusual jejunal dilatation (JD) that had less similarity than the RSS sequences (NT of 86.5% and AA of 93-93.1%). The phylogenetic analysis determined four groups. Group I had sequences from Korea. The second group included sequences from Korea, China, and Brazil (not included in this work). The third group had studied RSS sequences grouped with the ABU-P1 strain and sequences from China and the United States. Finally, the sequences from JD were clustered in a separate group with a bootstrap of 100%, a group that was denoted as group IV, and included sequences from China. RDP4 and SimPlot analysis showed one point of recombination with the sequences of group III ChPV in the JD sequences. Herein, we show that circulating strains of ChPV exhibit genetic differences in the VP1 gene in Brazilian chicken flocks. Nevertheless, more studies are needed to determine the probability of a new genetic group of ChPV based on the analysis of the complete genome.

BACKGROUND: Enterocytozoon bieneusi is the most common species found in humans. Although E. bieneusi has been investigated in humans, genotype profile of E. bieneusi is not known in Türkiye.
METHODS: In this study, we screened E. bieneusi in patients (n = 94) with different types of malignant solid tumors by Real Time PCR and then sequenced E. bieneusi positive samples. All cancer patients were undergoing chemotherapy and had diarrhea. Moreover, as control groups, we also screened E. bieneusi in patients with diarrhea (n = 50) and without diarrhea (n = 50).
RESULTS: Among all patients analyzed, 33 (17%) were found to be E. bieneusi-positive. As the patients were categorized, the molecular prevalence of E. bieneusi increased to 25.5% among cancer patients with diarrhea. However, the molecular prevalence of E. bieneusi was found to be lower in patients with presenting only diarrhea (8%) and patients without diarrhea (10%). The high molecular prevalence value detected among cancer patients with diarrhea was also statistically significant compared to other patient groups (P = 0.00112 and P = 0.0269). Among the 33 Real Time PCR positive samples, 10 of them were amplified by nested PCR and among these 10 samples, 6 of them were successfully genotyped. The phylogenetic tree showed the presence of D and Type IV which were also identified in stray cats living in İzmir in our previous study.
CONCLUSIONS: High molecular prevalence value indicates the importance of screening stool samples of cancer patients with diarrhea for E. bieneusi and genotyping results indicate that D and Type IV are circulating between humans and cats.

Listeria monocytogenes is a human pathogen that has the ability to cause listeriosis, a disease with possible fatal outcomes. The typical route of infection is ingestion of the bacteria with contaminated food. In this study, 13 virulence-associated genes were examined with PCR in the genomes of 153 L. monocytogenes isolates collected from meat products and processing environments in Poland. All isolates possessed genes from LIPI-1-hly, actA, plcA, plcB and mpl-as well as four internalins: inlA, inlB, inlC, inlJ. Invasion-associated protein iap, as well as genes prfA and sigB, encoding regulatory proteins, were also detected in all isolates. Gene flaA, encoding flagellin, was detected in 113 (74%) isolates. This was the only gene that was not detected in all isolates, as its presence is serotype-dependent. Gene actA showed polymorphism with longer and shorter variants in PCR amplicons. Two isolates were characterized by truncated inlB genes, lacking 141 bp in their sequence, which was confirmed by gene sequencing. All isolates were positive in hemolysis assays, proving the synthesis of functional PrfA and Hly proteins. Four genotypes of L. monocytogenes based on actA polymorphism and two genotypes based on inlB polymorphism were distinguished within the isolates\' collection.

Hypothyroidism is a common endocrine disorder whose prevalence increases with age. The disease manifests itself when the thyroid gland fails to produce sufficient thyroid hormones. The disorder includes cases of congenital hypothyroidism (CH), but most cases exhibit hormonal feedback dysregulation and destruction of the thyroid gland by autoantibodies. In this study, we sought to identify causal genes for hypothyroidism in large populations. The study used the UK-Biobank (UKB) database, reporting on 13,687 cases of European ancestry. We used GWAS compilation from Open Targets (OT) and tuned protocols focusing on genes and coding regions, along with complementary association methods of PWAS (proteome-based) and TWAS (transcriptome-based). Comparing summary statistics from numerous GWAS revealed a limited number of variants associated with thyroid development. The proteome-wide association study method identified 77 statistically significant genes, half of which are located within the Chr6-MHC locus and are enriched with autoimmunity-related genes. While coding GWAS and PWAS highlighted the centrality of immune-related genes, OT and transcriptome-wide association study mostly identified genes involved in thyroid developmental programs. We used independent populations from Finland (FinnGen) and the Taiwan cohort to validate the PWAS results. The higher prevalence in females relative to males is substantiated as the polygenic risk score prediction of hypothyroidism relied mostly from the female group genetics. Comparing results from OT, TWAS, and PWAS revealed the complementary facets of hypothyroidism\'s etiology. This study underscores the significance of synthesizing gene-phenotype association methods for this common, intricate disease. We propose that the integration of established association methods enhances interpretability and clinical utility.

We detected malaria vector Anopheles stephensi mosquitoes in the Al Hudaydah governorate in Yemen by using DNA sequencing. We report 2 cytochrome c oxidase subunit I haplotypes, 1 previously found in Ethiopia, Somalia, Djibouti, and Yemen. These findings provide insight into invasive An. stephensi mosquitoes in Yemen and their connection to East Africa.

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UNASSIGNED: Wild rodents are key hosts for Cryptosporidium transmission, yet there is a dearth of information regarding their infection status in the Inner Mongolian Autonomous Region and Liaoning Province of China. Therefore, the present study was conducted to determine the prevalence and genetic characteristics of Cryptosporidium among wild rodents residing in these two provinces.
UNASSIGNED: A total of 486 rodents were captured, and fresh feces were collected from each rodent\'s intestine for DNA extraction. Species identification of rodents was performed through PCR amplification of the vertebrate cytochrome b (cytb) gene. To detect the presence of Cryptosporidium in all fecal samples, PCR analysis and sequencing of the partial small subunit of the ribosomal RNA (rRNA) gene were performed.
UNASSIGNED: Four species of rodents were identified: Rattus norvegicus, Mus musculus, Apodemus agrarius, and Cricetulus barabensis. Positive results for Cryptosporidium were obtained for 9.2% (18/195), 6.6% (7/106), 5.6% (5/89), and 6.3% (6/96) of these rodents, respectively, with an average infection rate of 7.4% (36/486). The identification revealed the presence of five Cryptosporidium species, C. ubiquitum (n = 8), C. occultus (n = 5), C. muris (n = 2), C. viatorum (n = 1), and C. ratti (n = 1), along with two Cryptosporidium genotypes: Rat genotype III (n = 10) and Rat genotype IV (n = 9).
UNASSIGNED: Based on the molecular evidence presented, the wild rodents investigated were concurrently infected with zoonotic (C. muris, C. occultus, C. ubiquitum and C. viatorum) as well as rodent-adapted (C. ratti and Rat genotype III and IV) species/genotypes, actively participating in the transmission of cryptosporidiosis.