Visual genetic typing of glioma using proximity-anchored in situ spectral coding amplification.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.

Edwardsiella tarda in Tambaqui (Colossoma macropomum): A Pathogenicity, Antimicrobial Susceptibility, and Genetic Analysis of Brazilian Isolates.

Edwardsiella tarda is a crucial pathogenic bacterium in tropical aquaculture. This bacterium was recently isolated from tambaqui (Colossoma macropomum), a commercially important fish species in Brazil. This study assessed the antimicrobial susceptibility, pathogenicity, and genetic diversity of the tambaqui-derived E. tarda isolates. Fourteen bacterial isolates isolated from tambaqui were identified as E. tarda by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and dnaJ gene sequencing. Antimicrobial susceptibility tests were conducted against seven drugs using the disc diffusion assay. The pathogenicity test conducted by intraperitoneal injection of 2.4 × 107 colony-forming units (CFU) fish-1 of E. tarda (ED38-17) into tambaqui juveniles eventually revealed that neither clinical signs nor death were present. However, splenomegaly and whitish areas in the spleen and kidneys were observed. The histological investigation also revealed granulomatous splenitis, nephritis, and hepatitis occurring internally. Repetitive extragenic palindromic-PCR fingerprinting separated the 14 isolates into three genetic groups. The antibiogram revealed that all E. tarda isolates were wild-type (WT) to florfenicol (FLO), norfloxacin (NOR), neomycin (NEO), erythromycin (ERY), and oxytetracycline (OXY); however, some were non-wild-type to sulfamethoxazole/trimethoprim (7.1%) and amoxicillin (21.4%). Therefore, through experimental infection, E. tarda ED38-17 could induce pathogenic effects in C. macropomum. Additionally, three distinct genetic types were found, and the E. tarda isolates were WT to FLO, NOR, NEO, ERY, and OXY. These findings raise awareness of a bacteria causing unseen lesions, a pathogen that will potentially impact tambaqui aquaculture in the future.

Long-term acute infections during a bovine viral diarrhea virus (BVDV) outbreak in dairy farm from Galicia (NW Spain).

An observational study describes an outbreak of bovine viral diarrhea virus (BVDV) in a dairy herd in Spain. The herd was subjected to a voluntary control program. In a sampling carried out in June 2020, bulk tank milk antibody levels increased compared to the previous sampling. Additionally, serum samples from 4 young heifers also tested positive for antibodies. Since the results were consistent with a recent infection, we proceeded to detect possible persistently infected (PI) animals using antigen ELISA (on serum/ear-notch samples), following the program guidelines. From this moment on, 42 animals tested positive for BVDV antigen, of which 17 were under typical acute infection (AI), 13 were deemed as PI, and eight died early on the farm before having information to determine their status. The remaining 4 showed intriguing test results consistent with a long-term AI since they tested BVDV positive in at least two antigen tests more than 3 weeks apart. Thus, one animal was positive until 80 days of age in serum, and others even for longer periods in ear-notch samples, until they finally tested negative for BVDV. Based on these results, longer follow-up may be necessary in BVDV positive animals to accurately confirm persistent infection.

Polymorphic genetic markers and how they are associated with clinical and metabolic indicators of type 2 diabetes mellitus in the Kazakh population.

BACKGROUND: Type 2 diabetes mellitus is a serious public health problem worldwide. The aim of the study was to analyze the relationship of eight polymorphic gene variants with the development of clinical-metabolic rates of type 2 diabetes mellitus inside Kazakh population. MATERIALS AND METHODS: 139 patients with type 2 diabetes mellitus and 100 patients in the control group were examined. Genotyping of polymorphisms of candidate genes was carried out on a next generation QuantStudio 12 K Flex unit. RESULTS: Gene TCF7L2 locus rs7901695 and rs7903146, gene KCNQ1 locus rs2237892, rs7756992, and gene CDKAL1 locus rs7754840 demonstrated statistically significant associations with glucose metabolism, lipid profile and body mass index (BMI) in type 2 DM inside the population. Statistically significant difference in anthropometric and biochemical measures of rs17584499, rs4712523 and rs163184 has not been revealed. CONCLUSIONS: Genetic polymorphisms that influence pancreatic gland beta-cells insulin release and secretion associate with metabolic and anthropometric measures definitive for type 2 DM in Kazakh population.

Genotyping and Molecular Characterization of Classical Swine Fever Virus Isolated in China during 2016-2018.

Classical swine fever (CSF) is a highly contagious disease of swine caused by classical swine fever virus (CSFV). For decades the disease has been controlled in China by a modified live vaccine (C-strain) of genotype 1. The emergent genotype 2 strains have become predominant in China in the past years that are genetically distant from the vaccine strain. Here, we aimed to evaluate the current infectious status of CSF, and for this purpose 24 isolates of CSFV were identified from different areas of China during 2016-2018. Phylogenetic analysis of NS5B, E2 and full genome revealed that the new isolates were clustered into subgenotype 2.1d and 2.1b, while subgenotype 2.1d was predominant. Moreover, E2 and Erns displayed multiple variations in neutralizing epitope regions. Furthermore, the new isolates exhibited capacity to escape C-strain-derived antibody neutralization compared with the Shimen strain (genotype 1). Potential positive selection sites were identified in antigenic regions of E2 and Erns, which are related with antibody binding affinity. Recombination events were predicted in the new isolates with vaccine strains in the E2 gene region. In conclusion, the new isolates showed molecular variations and antigenic alterations, which provide evidence for the emergence of vaccine-escaping mutants and emphasize the need of updated strategies for CSF control.

Phenotypic and molecular characterisation of Sporothrix globosa of diverse origin from India.

Sporotrichosis is one of the neglected tropical diseases causing subcutaneous chronic granulomatous lesion by thermally dimorphic fungi belonging to Sporothrix species. Sporothrix brasiliensis, Sporothrix mexicana and Sporothrix globosa are the common pathogenic species. In Asian countries, S. globosa constitutes nearly 99.3% of all Sporothrix species. We studied 63 cases of sporotrichosis of geographically diverse origin from India and Sporothrix isolates were characterised for its growth in different media, temperatures, ability to assimilate sugars and antifungal susceptibility profile. Molecular characterization was performed by sequencing of the calmodulin (CAL), beta tubulin (BT) and translational elongation factor 1-alpha (TEF-1α) and typing by fluorescent amplified fragment length polymorphism (FAFLP). In patients who presented with fixed (49.2%), lymphocutaneous lesions (23.8%), in 26.9% the details were not known, none had systemic dissemination. All the isolates tested were Sporothrix globosa and that could grow up to 35 °C and unable to grow at and beyond 37 °C. The assimilation of sucrose, ribitol and raffinose helps in identifying S. globosa. Sequences of CAL or BT or TEF-1α can differentiate S. globosa from other species in the complex. FAFLP results exhibited low genetic diversity. No correlation was noted between genotypes and clinical presentation, or geographic distribution. Itraconazole, terbinafine and posaconazole showed good in vitro antifungal activity against S. globosa whereas fluconazole and micafungin had no activity. S. globosa of Indian origin is relatively less pathogenic than other pathogenic Sporothrix species as it does not cause systemic dissemination and in the diagnostic laboratory, incubation of the cultures below 37 °C is essential for effective isolation.

Serological and molecular investigation of B (A) blood group and its transfusion strategy: A case report / 中国输血杂志

【Objective】 To study the serological and genetic characteristics of a case of B(A) blood group. 【Methods】 Serological and genetic ABO blood group typing were used to analyze the ABO subtype and family inheritance of the probands and her 8 family members. The B(A) blood type sample was used as the blood recipient, and the B-type and O-type donors were selected for cross-matching using microcolumn gel anti-human globulin method to evaluate the blood transfusion strategy. 【Results】 5 out of 9 family blood samples were B(A) phenotype, carrying B(A)04 allele. Among them, 1 was B(A)04/O1 type, and 4 were B(A)04/B type. The primary blood matching of B(A) blood type samples with type B and O recipients were all negative. 【Conclusion】 A total of 5 cases of B(A)04 blood type were found in this family investigation, and there were differences in serological manifestations. Washed RBCs with B and O type can be used for B(A) blood type transfusion, and type B suspended RBCs can be considered in case of emergency.

Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue.

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis of rabies in an individual from the Dominican Republic using both IHC and the LN34 pan-lyssavirus real-time RT-PCR assay on formalin-fixed brain tissue. The LN34 assay generates a 165 bp amplicon and demonstrated higher sensitivity than traditional PCR. Multiple efforts to amplify nucleic acid fragments larger than 300 bp using conventional PCR were unsuccessful, probably due to RNA fragmentation. Sequences generated from the LN34 amplicon linked the case to the rabies virus (RABV) strain circulating in the Ouest Department of Haiti to the border region between Haiti and the Dominican Republic. Direct sequencing of the LN34 amplicon allowed rapid and low-cost rabies genetic typing.

The dwarf tapeworm Hymenolepis nana in pet rodents in Slovakia-epidemiological survey and genetic analysis.

Rodents are popular companion animals and are often kept as pets for children. However, they can be reservoirs of a variety of zoonotic pathogens. As little attention is being paid to the possibility of acquiring parasitic infections from pet rodents, the occurrence of Hymenolepis nana in rodents from pet shops and breeding clubs of Slovakia was surveyed, with parallel genetic analyses to type isolates from rodent species. In 2016-2018, pooled faecal samples from 119 boxes with 228 mice, 191 rats, 124 hamsters and 25 Mongolian gerbils were collected from 12 pet shops and 3 breeding clubs in five cities of eastern Slovakia. H. nana eggs were detected in 25 (21.0%) boxes. Animals from pet shops were infected more frequently (24.6% positive boxes) than those from breeding clubs (17.2%), without statistical significance. The highest prevalence was recorded in rats from pet shops, where 41.7% of boxes contained parasite eggs. Hamsters and mice in pet shops were also frequently infected; in 23.8% and 25% of boxes, respectively, H. nana eggs were observed. Prevalence in rats and hamsters from breeding clubs was lower, but in mice surpassed 40%. Nine samples with positive PCR products in any of the four DNA regions, mitochondrial cox1 and nuclear pmy, ITS1 and ITS2 targets, gave profiles characteristic of H. nana. The results imply the risk of zoonotic transmission of hymenolepiasis in Slovakia. Particular attention should be given to hygiene level maintained while keeping rodents. Furthermore, rodents intended for sale should be tested for parasites and then dewormed.

Genotyping of Pestivirus A (Bovine Viral Diarrhea Virus 1) detected in faeces and in other specimens of domestic and wild ruminants at the wildlife-livestock interface.

Pestiviruses are widespread in the world among ungulates and infect both domestic and wild animals causing severe economic losses in livestock. Bovine Viral Diarrhea Virus type 1 (BVDV-1), now re-designated as Pestivirus A, causes diseases mainly in cattle, while few data are available about infection in wild ruminants and about the role of these animals in viral maintenance and spread. In order to investigate BVDV-1 infection in domestic and wild ruminants, especially at the wildlife/livestock interface, bulk tank milk from dairy cattle and sheep and spleen from red deer, roe deer and fallow deer were analysed. Furthermore, faecal samples from Apennine chamois and from wild deer were evaluated as a suitable sample for detecting and genotyping pestiviruses. BVDV-1 RNA was found in all animal species tested but not sheep. Genotyping based on partial 5'UTR and Npro sequences detected BVDV-1a in samples from Apennine chamois, red deer, roe deer and pasture-raised cattle, while BVDV-1c was found in a faecal sample from Apennine chamois and in a spleen sample from roe deer. For the first time BVDV-1 RNA was found and genotyped from faecal samples of wild ruminants and of cattle. BVDV-1a detection in Apennine chamois, red deer, roe deer and pasture-raised cattle suggests that the eventuality of viral transmission at the wildlife/livestock interface should be carefully evaluated. BVDV subgenotype 1c was found for the first time in roe deer and Apennine chamois in Central Italy, therefore the epidemiological role of these animals and the viral ecology should be further investigated.

Genetic Characterization of Toxoplasma gondii from Wild Rodents in Sichuan Province, Southwestern China.

BACKGROUND: Wild rodents are the intermediate hosts of Toxoplasma gondii. The distribution of genetic diversity of T. gondii in wild rodents is of importance to understand the transmission of this parasite. This study aimed to genetically characterize T. gondii isolates from wild rodents in Sichuan province, southwestern China in 2013. METHODS: Genomic DNA was extracted from 10 g wild rodents' brain samples. Semi-nested PCR and multilocous PCR-RFLP technology were performed to examine genetic diversity of T. gondii isolates as described previously. RESULTS: Overall, 181 brain tissues of different wild rodents, including Eothenomys miletus (n=88), Crocidura attenuate (n=9), Rattus rattus sladeni (n=46), Mus musculus Linnaeus (n=6) and R. niviventer (n=32) were tested for T. gondii DNA, respectively. Six of them were positive for the T. gondii B1 gene by semi-nested PCR amplification, 4 showed complete genotyping results for all 11 polymorphic loci (SAG1, SAG2, alt. SAG2, SAG3, BTUB, GRA6, L358, PK1, C22-8, C29-2 and Apico) by PCR-RFLP, determined to represent a potential new genotype (http://toxodb.org/toxo/). CONCLUSION: These results documented genetic characterization of T. gondii in wild rodents from Sichuan province, and enriched the genetic diversity of T. gondii in China.

Prevalence of bovine viral diarrhea virus in dairy cattle herds in eastern China.

Bovine viral diarrhea virus (BVDV) is a worldwide spreading pestivirus affecting cattle and other ruminants; however, there have been few reports on epidemiologic investigation of BVDV in eastern China. In this study, bulk tank milk from 36 herds of dairy cattle in eastern China was submitted to serological investigations, 77.8% of herds was BVDV antibody positive. Individual animal status in two herds was further investigated collecting blood samples, the positive ratio was 49.74% and 24.64%, and the average positive ratio of calves, heifers, and lactating cows was 15.94%, 40.16%, and 41.7%, respectively. Moreover, clinical survey was carried out among 8170 dairy cattle from 36 herds, for diarrhea syndrome, respiratory problems and reproductive failure, and pathogens of all clinical cattle were further investigated. The results showed that BVDV was one of the main pathogen, which infected animals combining with various other viruses. Then, nine BVDV strains were isolated; phylogenetic analysis showed that BVDV subtypes currently circulating in eastern China were BVDV 1a and BVDV 1c. In addition, out of 377 cows tested, the 1.86% detected positive to the BVDV antigen. This study provided the foundation of further study on vaccination and control strategies of BVDV in eastern China.

ERIC-PCR Genotyping of Acinetobacter baumannii Isolated from Different Clinical Specimens.

BACKGROUND: Acinetobacter baumannii is a major cause of hospital care-acquired infections, and this bacterium poses a significant challenge to health care worldwide. At King Fahd Hospital of the University (KFHU), Al Khobar, Saudi Arabia, there had been a significant increase in the number of cases of A. baumannii infections. OBJECTIVE: The objective of this study was to determine the clonal relationship between A. baumannii collected from different specimens of patients admitted to KFHU using the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) fingerprinting method. MATERIALS AND METHODS: A. baumannii strains were isolated from a total of 59 specimens from inpatients admitted to KFHU between January and September 2014. These specimens were mainly collected from wound, rectal and throat swabs and transtracheal aspiration. ERIC-PCR fingerprinting was used to determine the clonal relationship between the different isolated strains. RESULTS: Using ERIC-PCR fingerprinting genotype analysis, 51 strains of A. baumannii were clustered into seven groups, while the remaining 8 were single strains. The genetic relatedness of A. baumannii isolated from admitted patients was high, indicating cross-transmission within the hospitalized patients. CONCLUSION: This study found that the increase in the incidence of A. baumannii in patients at KFHU was likely due to the spread of seven epidemic clones, thereby highlighting the need for intensifying the infection control measures to prevent nosocomial transmission of A. baumannii. These results also demonstrate that ERIC-PCR is a reliable and rapid method for studying the clonal similarity between A. baumannii isolated from different clinical specimens.

Tracking Listeria monocytogenes contamination and virulence-associated characteristics in the ready-to-eat meat-based food products industry according to the hygiene level.

Listeria monocytogenes isolates collected from final products and food contact surfaces of 10 ready-to-eat meat-based food products (RTEMP) producing industries were analyzed to relate their virulence-associated characteristics and genetic profiles with the hygiene assessment of those industries. Together with sample collection, an audit was performed to evaluate the implemented food safety management system and to investigate the specific audit requisites more associated to the occurrence of those L. monocytogenes serogroups frequently related with human disease. L. monocytogenes was present in 18% of the samples. The isolates (n=62) were serogrouped and detection of virulence-associated genes inlA, inlB, inlC and inlJ, and also plcA, hlyA, actA and iap was done by multiplex PCR. After this initial characterization, selected isolates (n=31) were submitted to antibiotic resistance testing by the disk diffusion method for the currently most used human and veterinary antibiotics and resistance was low. These isolates were also subtyped by pulsed-field gel electrophoresis. Genotyping and serogrouping of L. monocytogenes isolates revealed a genetically diverse population. Our data indicate that contamination of final products does not seem to be uniquely related to the sampled food surfaces. The occurrence of those L. monocytogenes serogroups more commonly associated with human disease in industries with a high hygienic audit classification could be the result of a previous identification of the pathogen, with an enforcement of the hygiene program without recognizing the real source of contamination. This reinforces the importance of a conjoined diagnosis using audit data and microbiological testing. Food safety management systems of those industries need improvement, particularly in cleaning and sanitizing operations, analytical control, preventive maintenance, personal hygiene and root cause analysis.

Isolation and Characterization of Enteroviruses from Clinical Samples.

Enterovirus infections are common in humans worldwide. Enteroviruses are excreted in feces during infection and can be detected from stool specimens by isolation in continuous laboratory cell lines. Characterization of enteroviruses is based on their antigenic and/or genetic properties.

Identification and molecular characterization of border disease virus (BDV) from sheep in India.

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5'-UTR, N(pro) and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N(pro) and entire region coding structural proteins showed that the N(pro) (168), C (100 aa), E(rns) (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.

Serum Amyloid A Protein Concentration in Blood is Influenced by Genetic Differences in the Cheetah (Acinonyx jubatus).

Systemic amyloid A (AA) amyloidosis is a major cause of morbidity and mortality among captive cheetahs. The self-aggregating AA protein responsible for this disease is a byproduct of serum amyloid A (SAA) protein degradation. Transcriptional induction of the SAA1 gene is dependent on both C/EBPß and NF-κB cis-acting elements within the promoter region. In cheetahs, 2 alleles exist for a single guanine nucleotide deletion in the putative NF-κB binding site. In this study, a novel genotyping assay was developed to screen for the alleles. The results show that the SAA1A (-97delG) allele is associated with decreased SAA protein concentrations in the serum of captive cheetahs (n = 58), suggesting genetic differences at this locus may be affecting AA amyloidosis prevalence. However, there was no significant difference in the frequency of the SAA1A (-97delG) allele between individuals confirmed AA amyloidosis positive versus AA amyloidosis negative at the time of necropsy (n = 48). Thus, even though there is evidence that having more copies of the SAA1A (-97delG) allele results in a potentially protective decrease in serum concentrations of SAA protein in captive cheetahs, genotype is not associated with this disease within the North American population. These results suggest that other factors are playing a more significant role in the pathogenesis of AA amyloidosis among captive cheetahs.

Tetragenococcus koreensis is part of the microbiota in a traditional Italian raw fermented sausage.

This study reports the isolation of Tetragenococcus koreensis, a bacterial species currently represented only by the type strain isolated from kimchi, from a raw fermented and ripened Italian sausage, Ventricina Vastese, all over the ripening period of five months. Rep-PCR genotyping showed that different T. koreensis strains, identified by sequencing of the 16S rRNA gene, were present in the same production batch. Tests on representative isolates showed intra-species physiological variability and the possession of phenotypic traits relevant for the production of fermented sausages, i.e. ability to grow at high salt concentrations, to induce some changes in the peptide profile of the culture medium and inability to produce histamine and tyramine, confirmed by the absence of the respective decarboxylase genes. Therefore the opportunity to further investigate the suitability of T. koreensis as a starter for fermented meat products was suggested.

Genotypic and phenotypic characterization of foodborne Geobacillus stearothermophilus.

Geobacillus stearothermophilus is the main thermophilic spore former involved in flat sour spoilage of canned foods. Three typing methods were tested and applied to differentiate strains at intra-species level: panC sequence analysis, REP-PCR and M13-PCR. panC gene was highly conserved within the studied strains, suggesting a low intra-specific diversity. This was supported by REP-PCR primary assays and M13-PCR results. M13-PCR profile analysis succeeded in differentiating six closely related groups (at 79% threshold similarity) among 127 strains from a range of spoiled canned food products and from different canneries. Phenotypic traits were investigated among 20 selected strains representing groups and origins. Ranges of growth under different temperatures (from 40 °C to 70 °C), pH (from 5.0 to 6.5), NaCl concentrations (from 1 to 5%) and sporulation conditions poorly differed between strains, but wet heat resistance of spores showed a 20-fold variation between strains. Furthermore, in this study, strains that belonged to the same M13-PCR genetic group did not share phenotypic characteristics or common origin. The work emphasizes a low diversity within the G. stearothermophilus species but data from this study may contribute to a better control of G. stearothermophilus spoilage in canned food.